Glucose-induced lipogenesis in pancreatic beta-cells is dependent on SREBP-1

DEFF Research Database (Denmark)

Sandberg, Maria B; Fridriksson, Jakob; Madsen, Lise

2005-01-01

High concentrations of glucose induce de novo fatty acid synthesis in pancreatic beta-cells and chronic exposure of elevated glucose and fatty acids synergize to induce accumulation of triglycerides, a phenomenon termed glucolipotoxicity. Here we investigate the role of sterol-regulatory element......, de novo fatty acid synthesis and lipid accumulation are induced primarily through sterol-regulatory elements (SREs) and not E-Boxes. Adenoviral expression of a dominant negative SREBP compromises glucose induction of some lipogenic genes and significantly reduces glucose-induction of de novo fatty...... acid synthesis. Thus, we demonstrate for the first time that SREBP activity is necessary for full glucose induction of de novo fatty acid synthesis in pancreatic beta-cells....

Effect on pancreatic beta cells and nerve cells by low let x-ray

Energy Technology Data Exchange (ETDEWEB)

Park, Kwang Hun [Dept. of Nuclear Medicine, Kyungbuk National University Hospital, Daegu (Korea, Republic of); Kim, Kgu Hwan [Dept. of Radiological Technology, Daegu health College, Daegu (Korea, Republic of)

2014-03-15

Cultured pancreatic beta cells and nerve cells, it is given normal condition of 10% FBS (fetal bovine serum), 11.1 mM glucose and hyperglycemia condition of 1% FBS, 30 mM glucose. For low LET X-ray irradiated with 0.5 Gy/hr dose-rate(total dose: 0.5 to 5 Gy). Survival rates were measured by MTT assay. When non irradiated, differentiated in the pancreatic beta cells experiment is hyperglycemia conditions survival rate compared to normal conditions survival rate seemed a small reduction. However increasing the total dose of X-ray, the survival rate of normal conditions decreased slightly compared to the survival rate of hyperglycemia conditions, the synergistic effect was drastically reduced. When non irradiated, undifferentiated in the nerve cells experiment is hyperglycemia conditions survival rate compared to normal conditions survival rate seemed a large reduction. As the cumulative dose of X-ray normal conditions and hyperglycemia were all relatively rapid cell death. But the rate of decreased survivals by almost parallel to the reduction proceed and it didn't show synergistic effect.

Pancreatic beta-cell overexpression of the glucagon receptor gene results in enhanced beta-cell function and mass

DEFF Research Database (Denmark)

Gelling, Richard W; Vuguin, Patricia M; Du, Xiu Quan

2009-01-01

in vivo, we generated mice overexpressing the Gcgr specifically on pancreatic beta-cells (RIP-Gcgr). In vivo and in vitro insulin secretion in response to glucagon and glucose was increased 1.7- to 3.9-fold in RIP-Gcgr mice compared with controls. Consistent with the observed increase in insulin release...

Trans fatty acids increase nitric oxide levels and pancreatic beta-cell necrosis in rats

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Kusmiyati Tjahjono DK

2013-04-01

Full Text Available Background The prevalence of diabetes in Indonesia is increasing due to various factors, including life style changes such as trans fatty acid (TFA intake. High TFA intake is known to be related to blood lipid profile changes resulting in cardiovascular disorders. This study was to identify the effect of TFA on nitric oxide (NO production and on necrosis of pancreatic beta cells. Methods A study of randomized pre-test post–test design with control group. Thirty Sprague Dawley rats were divided into 3 groups, i.e. group K (control, group P1 receiving a diet with 5% TFA, and P2 receiving 10% TFA. The intervention was performed for 8 weeks. NO level and pancreatic beta-cell necrosis were analyzed using Pearson’s chi square test. Results After 4 weeks of treatment there was no change in NO levels in group K, but increased NO in P2 (2.6-3.8 ìM. At 8 weeks after treatment, NO levels in groups P1 and P2 increased to 2.6-3.4 ìM and 4.2-14.3 ìM, respectively, while in group K only 2 rats had increased NO levels of 2.8-2.9 ìM. With Pearson’s chi-square test, there was a signifant difference in the proportions of necrotic pancreatic beta cells after 4 weeks and 8 weeks (p=0.000. No necrosis of beta cells was found in group K, mild necrosis in group P1 (1-25% and moderate necrosis in group P2 (26-50%. Conclusion TFA consumption significantly increases NO levels in Sprague Dawley rats and also results in moderate grades of necrosis of pancreatic beta cells.

Trans fatty acids increase nitric oxide levels and pancreatic beta-cell necrosis in rats

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Kusmiyati Tjahjono DK

2015-12-01

Full Text Available BACKGROUND The prevalence of diabetes in Indonesia is increasing due to various factors, including life style changes such as trans fatty acid (TFA intake. High TFA intake is known to be related to blood lipid profile changes resulting in cardiovascular disorders. This study was to identify the effect of TFA on nitric oxide (NO production and on necrosis of pancreatic beta cells. METHODS A study of randomized pre-test post–test design with control group. Thirty Sprague Dawley rats were divided into 3 groups, i.e. group K (control, group P1 receiving a diet with 5% TFA, and P2 receiving 10% TFA. The intervention was performed for 8 weeks. NO level and pancreatic beta-cell necrosis were analyzed using Pearson’s chi square test. RESULTS After 4 weeks of treatment there was no change in NO levels in group K, but increased NO in P2 (2.6-3.8 ìM. At 8 weeks after treatment, NO levels in groups P1 and P2 increased to 2.6-3.4 ìM and 4.2-14.3 ìM, respectively, while in group K only 2 rats had increased NO levels of 2.8-2.9 ìM. With Pearson’s chi-square test, there was a signifant difference in the proportions of necrotic pancreatic beta cells after 4 weeks and 8 weeks (p= 0.000. No necrosis of beta cells was found in group K, mild necrosis in group P1 (1-25% and moderate necrosis in group P2 (26-50%. CONCLUSION TFA consumption significantly increases NO levels in Sprague Dawley rats and also results in moderate grades of necrosis of pancreatic beta cells

Planar Cell Polarity Controls Pancreatic Beta Cell Differentiation and Glucose Homeostasis

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Cedric Cortijo

2012-12-01

Full Text Available Planar cell polarity (PCP refers to the collective orientation of cells within the epithelial plane. We show that progenitor cells forming the ducts of the embryonic pancreas express PCP proteins and exhibit an active PCP pathway. Planar polarity proteins are acquired at embryonic day 11.5 synchronously to apicobasal polarization of pancreas progenitors. Loss of function of the two PCP core components Celsr2 and Celsr3 shows that they control the differentiation of endocrine cells from polarized progenitors, with a prevalent effect on insulin-producing beta cells. This results in a decreased glucose clearance. Loss of Celsr2 and 3 leads to a reduction of Jun phosphorylation in progenitors, which, in turn, reduces beta cell differentiation from endocrine progenitors. These results highlight the importance of the PCP pathway in cell differentiation in vertebrates. In addition, they reveal that tridimensional organization and collective communication of cells are needed in the pancreatic epithelium in order to generate appropriate numbers of endocrine cells.

Role of growth factors in control of pancreatic beta cell mass: focus on betatrophin.

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Levitsky, Lynne L; Ardestani, Goli; Rhoads, David B

2014-08-01

Betatrophin is a newly described hormone, which potently stimulates beta cell replication in mice. This discovery has engendered great hope that it could prove clinically important in the treatment of type 1 and type 2 diabetes. Betatrophin, a 198-amino acid protein secreted by liver and adipose tissue, stimulates growth of pancreatic beta cell mass in insulin-resistant mice. Betatrophin has previously been named RIFL, lipasin, and ANGPLT8, and its salutory effects on lipid metabolism have been described in mouse and human studies. Serum betatrophin levels in humans correlate with improved adipose tissue lipid storage and lower serum triglyceride levels in the fed state, but do not correlate with insulin resistance or carbohydrate tolerance in humans. Betatrophin has not yet been shown to have an effect on beta cell replication in human pancreatic islets. Many endocrine and paracrine factors, of which betatrophin is the newest described, increase beta cell mass in murine models. None of these factors, including betatrophin, have displayed the same activity in clinical studies. This may reflect a profound species difference in beta cell regeneration pathways in mice and humans.

Expression of transient receptor potential ankyrin 1 (TRPA1 and its role in insulin release from rat pancreatic beta cells.

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De-Shou Cao

Full Text Available Several transient receptor potential (TRP channels are expressed in pancreatic beta cells and have been proposed to be involved in insulin secretion. However, the endogenous ligands for these channels are far from clear. Here, we demonstrate the expression of the transient receptor potential ankyrin 1 (TRPA1 ion channel in the pancreatic beta cells and its role in insulin release. TRPA1 is an attractive candidate for inducing insulin release because it is calcium permeable and is activated by molecules that are produced during oxidative glycolysis.Immunohistochemistry, RT-PCR, and Western blot techniques were used to determine the expression of TRPA1 channel. Ca²⁺ fluorescence imaging and electrophysiology (voltage- and current-clamp techniques were used to study the channel properties. TRPA1-mediated insulin release was determined using ELISA.TRPA1 is abundantly expressed in a rat pancreatic beta cell line and freshly isolated rat pancreatic beta cells, but not in pancreatic alpha cells. Activation of TRPA1 by allyl isothiocyanate (AITC, hydrogen peroxide (H₂O₂, 4-hydroxynonenal (4-HNE, and cyclopentenone prostaglandins (PGJ₂ and a novel agonist methylglyoxal (MG induces membrane current, depolarization, and Ca²⁺ influx leading to generation of action potentials in a pancreatic beta cell line and primary cultured pancreatic beta cells. Activation of TRPA1 by agonists stimulates insulin release in pancreatic beta cells that can be inhibited by TRPA1 antagonists such as HC030031 or AP-18 and by RNA interference. TRPA1-mediated insulin release is also observed in conditions of voltage-gated Na⁺ and Ca²⁺ channel blockade as well as ATP sensitive potassium (K(ATP channel activation.We propose that endogenous and exogenous ligands of TRPA1 cause Ca²⁺ influx and induce basal insulin release and that TRPA1-mediated depolarization acts synergistically with K(ATP channel blockade to facilitate insulin release.

Activation of the transcription factor carbohydrate-responsive element-binding protein by glucose leads to increased pancreatic beta cell differentiation in rats.

Science.gov (United States)

Soggia, A; Flosseau, K; Ravassard, P; Szinnai, G; Scharfmann, R; Guillemain, G

2012-10-01

Pancreatic cell development is a tightly controlled process. Although information is available regarding the mesodermal signals that control pancreatic development, little is known about the role of environmental factors such as nutrients, including glucose, on pancreatic development. We previously showed that glucose and its metabolism through the hexosamine biosynthesis pathway (HBP) promote pancreatic endocrine cell differentiation. Here, we analysed the role of the transcription factor carbohydrate-responsive element-binding protein (ChREBP) in this process. This transcription factor is activated by glucose, and has been recently described as a target of the HBP. We used an in vitro bioassay in which pancreatic endocrine and exocrine cells develop from rat embryonic pancreas in a way that mimics in vivo pancreatic development. Using this model, gain-of-function and loss-of-function experiments were undertaken. ChREBP was produced in the endocrine lineage during pancreatic development, its abundance increasing with differentiation. When rat embryonic pancreases were cultured in the presence of glucose or xylitol, the production of ChREBP targets was induced. Concomitantly, beta cell differentiation was enhanced. On the other hand, when embryonic pancreases were cultured with inhibitors decreasing ChREBP activity or an adenovirus producing a dominant-negative ChREBP, beta cell differentiation was reduced, indicating that ChREBP activity was necessary for proper beta cell differentiation. Interestingly, adenovirus producing a dominant-negative ChREBP also reduced the positive effect of N-acetylglucosamine, a substrate of the HBP acting on beta cell differentiation. Our work supports the idea that glucose, through the transcription factor ChREBP, controls beta cell differentiation from pancreatic progenitors.

Measuring phospholipase D activity in insulin-secreting pancreatic beta-cells and insulin-responsive muscle cells and adipocytes.

Science.gov (United States)

Cazzolli, Rosanna; Huang, Ping; Teng, Shuzhi; Hughes, William E

2009-01-01

Phospholipase D (PLD) is an enzyme producing phosphatidic acid and choline through hydrolysis of phosphatidylcholine. The enzyme has been identified as a member of a variety of signal transduction cascades and as a key regulator of numerous intracellular vesicle trafficking processes. A role for PLD in regulating glucose homeostasis is emerging as the enzyme has recently been identified in events regulating exocytosis of insulin from pancreatic beta-cells and also in insulin-stimulated glucose uptake through controlling GLUT4 vesicle exocytosis in muscle and adipose tissue. We present methodologies for assessing cellular PLD activity in secretagogue-stimulated insulin-secreting pancreatic beta-cells and also insulin-stimulated adipocyte and muscle cells, two of the principal insulin-responsive cell types controlling blood glucose levels.

Enhancing pancreatic Beta-cell regeneration in vivo with pioglitazone and alogliptin.

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Hao Yin

Full Text Available Pancreatic beta-cells retain limited ability to regenerate and proliferate after various physiologic triggers. Identifying therapies that are able to enhance beta-cell regeneration may therefore be useful for the treatment of both type 1 and type 2 diabetes.In this study we investigated endogenous and transplanted beta-cell regeneration by serially quantifying changes in bioluminescence from beta-cells from transgenic mice expressing firefly luciferase under the control of the mouse insulin I promoter. We tested the ability of pioglitazone and alogliptin, two drugs developed for the treatment of type 2 diabetes, to enhance beta-cell regeneration, and also defined the effect of the immunosuppression with rapamycin and tacrolimus on transplanted islet beta mass.Pioglitazone is a stimulator of nuclear receptor peroxisome proliferator-activated receptor gamma while alogliptin is a selective dipeptidyl peptidase IV inhibitor. Pioglitazone alone, or in combination with alogliptin, enhanced endogenous beta-cell regeneration in streptozotocin-treated mice, while alogliptin alone had modest effects. In a model of syngeneic islet transplantation, immunosuppression with rapamycin and tacrolimus induced an early loss of beta-cell mass, while treatment with insulin implants to maintain normoglycemia and pioglitazone plus alogliptin was able to partially promote beta-cell mass recovery.These data highlight the utility of bioluminescence for serially quantifying functional beta-cell mass in living mice. They also demonstrate the ability of pioglitazone, used either alone or in combination with alogliptin, to enhance regeneration of endogenous islet beta-cells as well as transplanted islets into recipients treated with rapamycin and tacrolimus.

Restoring Mitochondrial Function: A Small Molecule-mediated Approach to Enhance Glucose Stimulated Insulin Secretion in Cholesterol Accumulated Pancreatic beta cells

Science.gov (United States)

Asalla, Suman; Girada, Shravan Babu; Kuna, Ramya S.; Chowdhury, Debabrata; Kandagatla, Bhaskar; Oruganti, Srinivas; Bhadra, Utpal; Bhadra, Manika Pal; Kalivendi, Shasi Vardhan; Rao, Swetha Pavani; Row, Anupama; Ibrahim, A.; Ghosh, Partha Pratim; Mitra, Prasenjit

2016-06-01

Dyslipidemia, particularly the elevated serum cholesterol levels, aggravate the pathophysiology of type 2 diabetes. In the present study we explored the relationship between fasting blood sugar and serum lipid parameters in human volunteers which revealed a significant linear effect of serum cholesterol on fasting blood glucose. Short term feeding of cholesterol enriched diet to rodent model resulted in elevated serum cholesterol levels, cholesterol accumulation in pancreatic islets and hyperinsulinemia with modest increase in plasma glucose level. To explore the mechanism, we treated cultured BRIN-BD11 pancreatic beta cells with soluble cholesterol. Our data shows that cholesterol treatment of cultured pancreatic beta cells enhances total cellular cholesterol. While one hour cholesterol exposure enhances insulin exocytosis, overnight cholesterol accumulation in cultured pancreatic beta cells affects cellular respiration, and inhibits Glucose stimulated insulin secretion. We further report that (E)-4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl) hydrazono) ethyl) phenol (small molecule M1) prevents the cholesterol mediated blunting of cellular respiration and potentiates Glucose stimulated insulin secretion which was abolished in pancreatic beta cells on cholesterol accumulation.

EPConDB: a web resource for gene expression related to pancreatic development, beta-cell function and diabetes.

Science.gov (United States)

Mazzarelli, Joan M; Brestelli, John; Gorski, Regina K; Liu, Junmin; Manduchi, Elisabetta; Pinney, Deborah F; Schug, Jonathan; White, Peter; Kaestner, Klaus H; Stoeckert, Christian J

2007-01-01

EPConDB (http://www.cbil.upenn.edu/EPConDB) is a public web site that supports research in diabetes, pancreatic development and beta-cell function by providing information about genes expressed in cells of the pancreas. EPConDB displays expression profiles for individual genes and information about transcripts, promoter elements and transcription factor binding sites. Gene expression results are obtained from studies examining tissue expression, pancreatic development and growth, differentiation of insulin-producing cells, islet or beta-cell injury, and genetic models of impaired beta-cell function. The expression datasets are derived using different microarray platforms, including the BCBC PancChips and Affymetrix gene expression arrays. Other datasets include semi-quantitative RT-PCR and MPSS expression studies. For selected microarray studies, lists of differentially expressed genes, derived from PaGE analysis, are displayed on the site. EPConDB provides database queries and tools to examine the relationship between a gene, its transcriptional regulation, protein function and expression in pancreatic tissues.

Effect of iron on pancreatic beta cell function and insulin resistance ...

African Journals Online (AJOL)

Background: Increase in total body iron store has been reported in the aetiology and development of diabetes mellitus. The effect of iron supplementation in female with respect to the incidence of diabetes mellitus was investigated on the pancreatic beta cell function and insulin resistance in normal female rats. Methods: ...

Maternal obesity accelerates fetal pancreatic beta-cell but not alpha-cell development in sheep: prenatal consequences.

Science.gov (United States)

Ford, Stephen P; Zhang, Liren; Zhu, Meijun; Miller, Myrna M; Smith, Derek T; Hess, Bret W; Moss, Gary E; Nathanielsz, Peter W; Nijland, Mark J

2009-09-01

Maternal obesity affects offspring weight, body composition, and organ function, increasing diabetes and metabolic syndrome risk. We determined effects of maternal obesity and a high-energy diet on fetal pancreatic development. Sixty days prior to breeding, ewes were assigned to control [100% of National Research Council (NRC) recommendations] or obesogenic (OB; 150% NRC) diets. At 75 days gestation, OB ewes exhibited elevated insulin-to-glucose ratios at rest and during a glucose tolerance test, demonstrating insulin resistance compared with control ewes. In fetal studies, ewes ate their respective diets from 60 days before to 75 days after conception when animals were euthanized under general anesthesia. OB and control ewes increased in body weight by approximately 43% and approximately 6%, respectively, from diet initiation until necropsy. Although all organs were heavier in fetuses from OB ewes, only pancreatic weight increased as a percentage of fetal weight. Blood glucose, insulin, and cortisol were elevated in OB ewes and fetuses on day 75. Insulin-positive cells per unit pancreatic area were 50% greater in fetuses from OB ewes as a result of increased beta-cell mitoses rather than decreased programmed cell death. Lambs of OB ewes were born earlier but weighed the same as control lambs; however, their crown-to-rump length was reduced, and their fat mass was increased. We conclude that increased systemic insulin in fetuses from OB ewes results from increased glucose exposure and/or cortisol-induced accelerated fetal beta-cell maturation and may contribute to premature beta-cell function loss and predisposition to obesity and metabolic disease in offspring.

The effect of smoking cessation pharmacotherapies on pancreatic beta cell function

International Nuclear Information System (INIS)

Woynillowicz, Amanda K.; Raha, Sandeep; Nicholson, Catherine J.; Holloway, Alison C.

2012-01-01

The goal of our study was to evaluate whether drugs currently used for smoking cessation (i.e., nicotine replacement therapy, varenicline [a partial agonist at nicotinic acetylcholine receptors (nAChR)] and bupropion [which acts in part as a nAChR antagonist]) can affect beta cell function and determine the mechanism(s) of this effect. INS-1E cells, a rat beta cell line, were treated with nicotine, varenicline and bupropion to determine their effects on beta cell function, mitochondrial electron transport chain enzyme activity and cellular/oxidative stress. Treatment of INS-1E cells with equimolar concentrations (1 μM) of three test compounds resulted in an ablation of normal glucose-stimulated insulin secretion by the cells. This disruption of normal beta cell function was associated with mitochondrial dysfunction since all three compounds tested significantly decreased the activity of mitochondrial electron transport chain enzyme activity. These results raise the possibility that the currently available smoking cessation pharmacotherapies may also have adverse effects on beta cell function and thus glycemic control in vivo. Therefore whether or not the use of nicotine replacement therapy, varenicline and bupropion can cause endocrine changes which are consistent with impaired pancreatic function warrants further investigation. -- Highlights: ► Smoking cessation drugs have the potential to disrupt beta cell function in vitro. ► The effects of nicotine, varenicline and bupropion are similar. ► The impaired beta cell function is mediated by mitochondrial dysfunction. ► If similar effects are seen in vivo, these drugs may increase the risk of diabetes.

The effect of smoking cessation pharmacotherapies on pancreatic beta cell function

Energy Technology Data Exchange (ETDEWEB)

Woynillowicz, Amanda K. [Department of Obstetrics and Gynecology, McMaster University, Hamilton, ON, Canada L8N 3Z5 (Canada); Raha, Sandeep [Department of Pediatrics, McMaster University, Hamilton, ON, Canada L8N 3Z5 (Canada); Nicholson, Catherine J. [Department of Obstetrics and Gynecology, McMaster University, Hamilton, ON, Canada L8N 3Z5 (Canada); Holloway, Alison C., E-mail: hollow@mcmaster.ca [Department of Obstetrics and Gynecology, McMaster University, Hamilton, ON, Canada L8N 3Z5 (Canada)

2012-11-15

The goal of our study was to evaluate whether drugs currently used for smoking cessation (i.e., nicotine replacement therapy, varenicline [a partial agonist at nicotinic acetylcholine receptors (nAChR)] and bupropion [which acts in part as a nAChR antagonist]) can affect beta cell function and determine the mechanism(s) of this effect. INS-1E cells, a rat beta cell line, were treated with nicotine, varenicline and bupropion to determine their effects on beta cell function, mitochondrial electron transport chain enzyme activity and cellular/oxidative stress. Treatment of INS-1E cells with equimolar concentrations (1 μM) of three test compounds resulted in an ablation of normal glucose-stimulated insulin secretion by the cells. This disruption of normal beta cell function was associated with mitochondrial dysfunction since all three compounds tested significantly decreased the activity of mitochondrial electron transport chain enzyme activity. These results raise the possibility that the currently available smoking cessation pharmacotherapies may also have adverse effects on beta cell function and thus glycemic control in vivo. Therefore whether or not the use of nicotine replacement therapy, varenicline and bupropion can cause endocrine changes which are consistent with impaired pancreatic function warrants further investigation. -- Highlights: ► Smoking cessation drugs have the potential to disrupt beta cell function in vitro. ► The effects of nicotine, varenicline and bupropion are similar. ► The impaired beta cell function is mediated by mitochondrial dysfunction. ► If similar effects are seen in vivo, these drugs may increase the risk of diabetes.

Regulation of Pancreatic Beta Cell Stimulus-Secretion Coupling by microRNAs

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Jonathan L. S. Esguerra

2014-11-01

Full Text Available Increased blood glucose after a meal is countered by the subsequent increased release of the hypoglycemic hormone insulin from the pancreatic beta cells. The cascade of molecular events encompassing the initial sensing and transport of glucose into the beta cell, culminating with the exocytosis of the insulin large dense core granules (LDCVs is termed “stimulus-secretion coupling.” Impairment in any of the relevant processes leads to insufficient insulin release, which contributes to the development of type 2 diabetes (T2D. The fate of the beta cell, when exposed to environmental triggers of the disease, is determined by the possibility to adapt to the new situation by regulation of gene expression. As established factors of post-transcriptional regulation, microRNAs (miRNAs are well-recognized mediators of beta cell plasticity and adaptation. Here, we put focus on the importance of comprehending the transcriptional regulation of miRNAs, and how miRNAs are implicated in stimulus-secretion coupling, specifically those influencing the late stages of insulin secretion. We suggest that efficient beta cell adaptation requires an optimal balance between transcriptional regulation of miRNAs themselves, and miRNA-dependent gene regulation. The increased knowledge of the beta cell transcriptional network inclusive of non-coding RNAs such as miRNAs is essential in identifying novel targets for the treatment of T2D.

Effect of prolonged exposure to sublethal concentrations of DDT and DDE on protein expression in human pancreatic beta cells

Energy Technology Data Exchange (ETDEWEB)

Pavlikova, Nela, E-mail: nela.pavlikova@lf3.cuni.cz [Department of Cell and Molecular Biology, Third Faculty of Medicine, Charles University, Prague (Czech Republic); Smetana, Pavel [Department of Cell and Molecular Biology, Third Faculty of Medicine, Charles University, Prague (Czech Republic); Halada, Petr [Laboratory of Molecular Structure Characterization, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague (Czech Republic); Kovar, Jan [Department of Cell and Molecular Biology, Third Faculty of Medicine, Charles University, Prague (Czech Republic)

2015-10-15

Pollution of the environment represents one of less explored potential reasons for the worldwide epidemic of type 2 diabetes. One of the most prevalent organochlorine pollutants remains the pesticide DDT and its degradation product DDE. Despite some epidemiologic correlations between levels of DDT and DDE in human organism and the prevalence of diabetes, there is almost no information about the exact targets of these compounds inside pancreatic beta cells. To detect functional areas of pancreatic beta cells that could be affected by exposure to DDT and DDE, we analyzed changes in protein expression in the NES2Y human pancreatic beta cell line exposed to three sublethal concentrations (0.1 μM, 1 μM, 10 μM) of DDT and DDE for 1 month. Protein separation and identification was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectrometry. With these techniques, four proteins were found downregulated after exposure to 10 μM DDT: three cytoskeletal proteins (cytokeratin 8, cytokeratin 18 and actin) and one protein involved in glycolysis (alpha-enolase). Two proteins were downregulated after exposure to 10 μM DDE: cytokeratin 18 and heterogenous nuclear ribonucleoprotein H1 (HNRH1). These changes correlate with previously described effects of other stress conditions (e.g. exposure to palmitate, hyperglycemia, imidazoline derivative, and cytokines) on protein expression in pancreatic beta cells. We conclude that cytoskeletal proteins and their processing, glucose metabolism, and mRNA processing may represent targets affected by exposure to conditions hostile to pancreatic beta cells, including exposure to DDT and DDE. - Highlights: • Epidemiologic studies connect pollution with incidence of diabetes mellitus. • We explored the effect of DDT and DDE on protein expression in the NES2Y pancreatic beta cell line. • One month exposure to three sublethal concentrations of DDT and DDE was employed. • Expression of alpha-enolase, actin

Effect of prolonged exposure to sublethal concentrations of DDT and DDE on protein expression in human pancreatic beta cells

International Nuclear Information System (INIS)

Pavlikova, Nela; Smetana, Pavel; Halada, Petr; Kovar, Jan

2015-01-01

Pollution of the environment represents one of less explored potential reasons for the worldwide epidemic of type 2 diabetes. One of the most prevalent organochlorine pollutants remains the pesticide DDT and its degradation product DDE. Despite some epidemiologic correlations between levels of DDT and DDE in human organism and the prevalence of diabetes, there is almost no information about the exact targets of these compounds inside pancreatic beta cells. To detect functional areas of pancreatic beta cells that could be affected by exposure to DDT and DDE, we analyzed changes in protein expression in the NES2Y human pancreatic beta cell line exposed to three sublethal concentrations (0.1 μM, 1 μM, 10 μM) of DDT and DDE for 1 month. Protein separation and identification was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectrometry. With these techniques, four proteins were found downregulated after exposure to 10 μM DDT: three cytoskeletal proteins (cytokeratin 8, cytokeratin 18 and actin) and one protein involved in glycolysis (alpha-enolase). Two proteins were downregulated after exposure to 10 μM DDE: cytokeratin 18 and heterogenous nuclear ribonucleoprotein H1 (HNRH1). These changes correlate with previously described effects of other stress conditions (e.g. exposure to palmitate, hyperglycemia, imidazoline derivative, and cytokines) on protein expression in pancreatic beta cells. We conclude that cytoskeletal proteins and their processing, glucose metabolism, and mRNA processing may represent targets affected by exposure to conditions hostile to pancreatic beta cells, including exposure to DDT and DDE. - Highlights: • Epidemiologic studies connect pollution with incidence of diabetes mellitus. • We explored the effect of DDT and DDE on protein expression in the NES2Y pancreatic beta cell line. • One month exposure to three sublethal concentrations of DDT and DDE was employed. • Expression of alpha-enolase, actin

Chronic antidiabetic sulfonylureas in vivo: reversible effects on mouse pancreatic beta-cells.

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Maria Sara Remedi

2008-10-01

Full Text Available Pancreatic beta-cell ATP-sensitive potassium (K ATP channels are critical links between nutrient metabolism and insulin secretion. In humans, reduced or absent beta-cell K ATP channel activity resulting from loss-of-function K ATP mutations induces insulin hypersecretion. Mice with reduced K ATP channel activity also demonstrate hyperinsulinism, but mice with complete loss of K ATP channels (K ATP knockout mice show an unexpected insulin undersecretory phenotype. Therefore we have proposed an "inverse U" hypothesis to explain the response to enhanced excitability, in which excessive hyperexcitability drives beta-cells to insulin secretory failure without cell death. Many patients with type 2 diabetes treated with antidiabetic sulfonylureas (which inhibit K ATP activity and thereby enhance insulin secretion show long-term insulin secretory failure, which we further suggest might reflect a similar progression.To test the above hypotheses, and to mechanistically investigate the consequences of prolonged hyperexcitability in vivo, we used a novel approach of implanting mice with slow-release sulfonylurea (glibenclamide pellets, to chronically inhibit beta-cell K ATP channels. Glibenclamide-implanted wild-type mice became progressively and consistently diabetic, with significantly (p < 0.05 reduced insulin secretion in response to glucose. After 1 wk of treatment, these mice were as glucose intolerant as adult K ATP knockout mice, and reduction of secretory capacity in freshly isolated islets from implanted animals was as significant (p < 0.05 as those from K ATP knockout animals. However, secretory capacity was fully restored in islets from sulfonylurea-treated mice within hours of drug washout and in vivo within 1 mo after glibenclamide treatment was terminated. Pancreatic immunostaining showed normal islet size and alpha-/beta-cell distribution within the islet, and TUNEL staining showed no evidence of apoptosis.These results demonstrate that

Lipotoxicity Mediated Cell Dysfunction and Death Involves Lysosomal Membrane Permeabilization and Cathepsin L Activity

Science.gov (United States)

Almaguel, Frankis G.; Liu, Jo-Wen; Pacheco, Fabio J.; De Leon, Daisy; Casiano, Carlos A.; De Leon, Marino

2010-01-01

Lipotoxicity, which is triggered when cells are exposed to elevated levels of free fatty acids, involves cell dysfunction and apoptosis and is emerging as an underlying factor contributing to various pathological conditions including disorders of the central nervous system and diabetes. We have shown that palmitic acid (PA)-induced lipotoxicity (PA-LTx) in nerve growth factor-differentiated PC12 (NGFDPC12) cells is linked to an augmented state of cellular oxidative stress (ASCOS) and apoptosis, and that these events are inhibited by docosahexanoic acid (DHA). The mechanisms of PA-LTx in nerve cells are not well understood, but our previous findings indicate that it involves ROS generation, mitochondrial membrane permeabilization (MMP), and caspase activation. The present study used nerve growth factor differentiated PC12 cells (NGFDPC12 cells) and found that lysosomal membrane permeabilization (LMP) is an early event during PA-induced lipotoxicity that precedes MMP and apoptosis. Cathepsin L, but not cathepsin B, is an important contributor in this process since its pharmacological inhibition significantly attenuated LMP, MMP, and apoptosis. In addition, co-treatment of NGFDPC12 cells undergoing lipotoxicity with DHA significantly reduced LMP, suggesting that DHA acts by antagonizing upstream signals leading to lysosomal dysfunction. These results suggest that LMP is a key early mediator of lipotoxicity, and underscore the value of interventions targeting upstream signals leading to LMP for the treatment of pathological conditions associated with lipotoxicity. PMID:20043885

The zinc transporter ZNT3 co-localizes with insulin in INS-1E pancreatic beta cells and influences cell survival, insulin secretion capacity, and ZNT8 expression

DEFF Research Database (Denmark)

Smidt, Kamille; Larsen, Agnete; Brønden, Andreas

2016-01-01

Zinc trafficking in pancreatic beta cells is tightly regulated by zinc transporting (ZNTs) proteins. The role of different ZNTs in the beta cells is currently being clarified. ZNT8 transports zinc into insulin granules and is critical for a correct insulin crystallization and storage in the granu......Zinc trafficking in pancreatic beta cells is tightly regulated by zinc transporting (ZNTs) proteins. The role of different ZNTs in the beta cells is currently being clarified. ZNT8 transports zinc into insulin granules and is critical for a correct insulin crystallization and storage...

Effect of prolonged exposure to sublethal concentrations of DDT and DDE on protein expression in human pancreatic beta cells.

Science.gov (United States)

Pavlikova, Nela; Smetana, Pavel; Halada, Petr; Kovar, Jan

2015-10-01

Pollution of the environment represents one of less explored potential reasons for the worldwide epidemic of type 2 diabetes. One of the most prevalent organochlorine pollutants remains the pesticide DDT and its degradation product DDE. Despite some epidemiologic correlations between levels of DDT and DDE in human organism and the prevalence of diabetes, there is almost no information about the exact targets of these compounds inside pancreatic beta cells. To detect functional areas of pancreatic beta cells that could be affected by exposure to DDT and DDE, we analyzed changes in protein expression in the NES2Y human pancreatic beta cell line exposed to three sublethal concentrations (0.1 μM, 1 μM, 10 μM) of DDT and DDE for 1 month. Protein separation and identification was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectrometry. With these techniques, four proteins were found downregulated after exposure to 10 μM DDT: three cytoskeletal proteins (cytokeratin 8, cytokeratin 18 and actin) and one protein involved in glycolysis (alpha-enolase). Two proteins were downregulated after exposure to 10 μM DDE: cytokeratin 18 and heterogenous nuclear ribonucleoprotein H1 (HNRH1). These changes correlate with previously described effects of other stress conditions (e.g. exposure to palmitate, hyperglycemia, imidazoline derivative, and cytokines) on protein expression in pancreatic beta cells. We conclude that cytoskeletal proteins and their processing, glucose metabolism, and mRNA processing may represent targets affected by exposure to conditions hostile to pancreatic beta cells, including exposure to DDT and DDE. Copyright © 2015 Elsevier Inc. All rights reserved.

Drp1 guarding of the mitochondrial network is important for glucose-stimulated insulin secretion in pancreatic beta cells

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Reinhardt, Florian; Schultz, Julia; Waterstradt, Rica; Baltrusch, Simone, E-mail: simone.baltrusch@med.uni-rostock.de

2016-06-10

Mitochondria form a tubular network in mammalian cells, and the mitochondrial life cycle is determined by fission, fusion and autophagy. Dynamin-related protein 1 (Drp1) has a pivotal role in these processes because it alone is able to constrict mitochondria. However, the regulation and function of Drp1 have been shown to vary between cell types. Mitochondrial morphology affects mitochondrial metabolism and function. In pancreatic beta cells mitochondrial metabolism is a key component of the glucose-induced cascade of insulin secretion. The goal of the present study was to investigate the action of Drp1 in pancreatic beta cells. For this purpose Drp1 was down-regulated by means of shDrp1 in insulin-secreting INS1 cells and mouse pancreatic islets. In INS1 cells reduced Drp1 expression resulted in diminished expression of proteins regulating mitochondrial fusion, namely mitofusin 1 and 2, and optic atrophy protein 1. Diminished mitochondrial dynamics can therefore be assumed. After down-regulation of Drp1 in INS1 cells and spread mouse islets the initially homogenous mitochondrial network characterised by a moderate level of interconnections shifted towards high heterogeneity with elongated, clustered and looped mitochondria. These morphological changes were found to correlate directly with functional alterations. Mitochondrial membrane potential and ATP generation were significantly reduced in INS1 cells after Drp1down-regulation. Finally, a significant loss of glucose-stimulated insulin secretion was demonstrated in INS1 cells and mouse pancreatic islets. In conclusion, Drp1 expression is important in pancreatic beta cells to maintain the regulation of insulin secretion. -- Highlights: •Down-regulation of Drp1 in INS1 cells reduces mitochondrial fusion protein expression. •Mitochondrial membrane potential in INS1 cells is diminished after Drp1 down-regulation. •Mitochondria become elongated after down-regulation of Drp1 in beta cells. •Down-regulation of

Insulin-like Growth Factor 1 Regulates the Expression of ATP-Binding Cassette Transporter A1 in Pancreatic Beta Cells.

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Lyu, J; Imachi, H; Iwama, H; Zhang, H; Murao, K

2016-05-01

ATP-binding cassette transporter A1 (ABCA1) in pancreatic beta cells influences insulin secretion and cholesterol homeostasis. The present study investigates whether insulin-like growth factor 1 (IGF-1), which mediates stimulation of ABCA1 gene expression, could also interfere with the phosphatidylinositol 3-kinase (PI3-K) cascade.ABCA1 expression was examined by real-time polymerase chain reaction (PCR), Western blot analysis, and a reporter gene assay in rat insulin-secreting INS-1 cells incubated with IGF-1. The binding of forkhead box O1 (FoxO1) protein to the ABCA1 promoter was assessed by a chromatin immunoprecipitation (ChIP) assay. ABCA1 protein levels increased in response to rising concentrations of IGF-1. Real-time PCR analysis showed a significant increase in ABCA1 mRNA expression. However, both effects were suppressed after silencing the IGF-1 receptor. In parallel with its effect on endogenous ABCA1 mRNA levels, IGF-1 induced the activity of a reporter construct containing the ABCA1 promoter, while it was abrogated by LY294002, a specific inhibitor of PI3-K. Constitutively active Akt stimulated activity of the ABCA1 promoter, and a dominant-negative mutant of Akt or mutagenesis of the FoxO1 response element in the ABCA1 promoter abolished the ability of IGF-1 to stimulate promoter activity. A ChIP assay showed that FoxO1 mediated its transcriptional activity by directly binding to the ABCA1 promoter region. The knockdown of FoxO1 disrupted the effect of IGF-1 on ABCA1 expression. Furthermore, IGF-1 promoted cholesterol efflux and reduced the pancreatic lipotoxicity. These results demonstrate that the PI3-K/Akt/FoxO1 pathway contributes to the regulation of ABCA1 expression in response to IGF-1 stimulation. © Georg Thieme Verlag KG Stuttgart · New York.

Curcumin attenuates palmitate-induced apoptosis in MIN6 pancreatic β-cells through PI3K/Akt/FoxO1 and mitochondrial survival pathways.

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Hao, Feng; Kang, Jinsen; Cao, Yajun; Fan, Shengjun; Yang, Haopeng; An, Yu; Pan, Yan; Tie, Lu; Li, Xuejun

2015-11-01

Lipotoxicity plays a vital role in development and progression of type 2 diabetes. Prolonged elevation of free fatty acids especially the palmitate leads to pancreatic β-cell dysfunction and apoptosis. Curcumin (diferuloylmethane), a polyphenol from the curry spice turmeric, is considered to be a broadly cytoprotective agent. The present study was designed to determine the protective effect of curcumin on palmitate-induced apoptosis in β-cells and investigate underlying mechanisms. Our results showed that curcumin improved cell viability and enhanced glucose-induced insulin secretory function in MIN6 pancreatic β-cells. Palmitate incubation evoked chromatin condensation, DNA nick end labeling and activation of caspase-3 and -9. Curcumin treatment inhibited palmitate-induced apoptosis, relieved mitochondrial depolarization and up-regulated Bcl-2/Bax ratio. Palmitate induced the generation of reactive oxygen species and inhibited activities of antioxidant enzymes, which could be neutralized by curcumin treatment. Moreover, curcumin could promote rapid phosphorylation of Akt and nuclear exclusion of FoxO1 in MIN6 cells under lipotoxic condition. Phosphatidylinositol 3-kinase and Akt specific inhibitors abolished the anti-lipotoxic effect of curcumin and stimulated FoxO1 nuclear translocation. These findings suggested that curcumin protected MIN6 pancreatic β-Cells against apoptosis through activation of Akt, inhibition of nuclear translocation of FoxO1 and mitochondrial survival pathway.

Intracellular serotonin modulates insulin secretion from pancreatic beta-cells by protein serotonylation.

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Nils Paulmann

2009-10-01

Full Text Available While serotonin (5-HT co-localization with insulin in granules of pancreatic beta-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is still unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph1-/- and 5-HT to show that intracellular 5-HT regulates insulin secretion. We found that these mice are diabetic and have an impaired insulin secretion due to the lack of 5-HT in the pancreas. The pharmacological restoration of peripheral 5-HT levels rescued the impaired insulin secretion in vivo. These findings were further evidenced by patch clamp experiments with isolated Tph1-/- beta-cells, which clearly showed that the secretory defect is downstream of Ca(2+-signaling and can be rescued by direct intracellular application of 5-HT via the clamp pipette. In elucidating the underlying mechanism further, we demonstrate the covalent coupling of 5-HT by transglutaminases during insulin exocytosis to two key players in insulin secretion, the small GTPases Rab3a and Rab27a. This renders them constitutively active in a receptor-independent signaling mechanism we have recently termed serotonylation. Concordantly, an inhibition of such activating serotonylation in beta-cells abates insulin secretion. We also observed inactivation of serotonylated Rab3a by enhanced proteasomal degradation, which is in line with the inactivation of other serotonylated GTPases. Our results demonstrate that 5-HT regulates insulin secretion by serotonylation of GTPases within pancreatic beta-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.

Cytoprotective effect of kaempferol against palmitic acid-induced pancreatic β-cell death through modulation of autophagy via AMPK/mTOR signaling pathway.

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Varshney, Ritu; Gupta, Sumeet; Roy, Partha

2017-06-15

Lipotoxicity of pancreatic β-cells is the pathological manifestation of obesity-linked type II diabetes. We intended to determine the cytoprotective effect of kaempferol on pancreatic β-cells undergoing apoptosis in palmitic acid (PA)-stressed condition. The data showed that kaempferol treatment increased cell viability and anti-apoptotic activity in PA-stressed RIN-5F cells and murine pancreatic islets. Furthermore, kaempferol's ability to instigate autophagy was illustrated by MDC-LysoTracker red staining and TEM analysis which corroborated well with the observed increase in LC3 puncta and LC3-II protein expressions along with the concomitant decline in p62 expression. Apart from this, the data showed that kaempferol up/down-regulates AMPK/mTOR phosphorylation respectively. Subsequently, upon inhibition of AMPK phosphorylation by AMPK inhibitors, kaempferol-mediated autophagy was abolished which further led to the decline in β-cell survival. Such observations collectively lead to the conclusion that, kaempferol exerts its cytoprotective role against lipotoxicity by activation of autophagy via AMPK/mTOR pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

Activation of the Wnt/β-catenin pathway in pancreatic beta cells during the compensatory islet hyperplasia in prediabetic mice

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Maschio, D.A.; Oliveira, R.B.; Santos, M.R.; Carvalho, C.P.F.; Barbosa-Sampaio, H.C.L.; Collares-Buzato, C.B.

2016-01-01

The Wnt/β-catenin signaling pathway, also known as the canonical Wnt pathway, plays a role in cell proliferation and differentiation in several tissues/organs. It has been recently described in humans a relationship between type 2 diabetes (T2DM) and mutation in the gene encoding the transcription factor TCF7L2 associated to the Wnt/β-catenin pathway. In the present study, we demonstrated that hyperplastic pancreatic islets from prediabetic mice fed a high-fat diet (HFD) for 60 d displayed nuclear translocation of active β-catenin associated with significant increases in protein content and gene expression of β-catenin as well as of cyclins D1, D2 and c-Myc (target genes of the Wnt pathway) but not of Tcf7l2 (the transcription factor). Meanwhile, these alterations were not observed in pancreatic islets from 30 d HFD-fed mice, that do not display significant beta cell hyperplasia. These data suggest that the Wnt/β-catenin pathway is activated in pancreatic islets during prediabetes and may play a role in the induction of the compensatory beta cell hyperplasia observed at early phase of T2DM. - Highlights: • Exposure to high-fat diet for 60 days induced prediabetes and beta cell mass expansion. • Hyperplastic pancreatic islets displayed nuclear translocation of active β-catenin. • Hyperplastic islets showed increased expression of target genes of the Wnt/β-catenin pathway. • Wnt/β-catenin pathway is activated during compensatory beta cell hyperplasia in mice.

In silico identification of NF-kappaB-regulated genes in pancreatic beta-cells

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Eizirik Decio L

2007-02-01

Full Text Available Abstract Background Pancreatic beta-cells are the target of an autoimmune attack in type 1 diabetes mellitus (T1DM. This is mediated in part by cytokines, such as interleukin (IL-1β and interferon (IFN-γ. These cytokines modify the expression of hundreds of genes, leading to beta-cell dysfunction and death by apoptosis. Several of these cytokine-induced genes are potentially regulated by the IL-1β-activated transcription factor (TF nuclear factor (NF-κB, and previous studies by our group have shown that cytokine-induced NF-κB activation is pro-apoptotic in beta-cells. To identify NF-κB-regulated gene networks in beta-cells we presently used a discriminant analysis-based approach to predict NF-κB responding genes on the basis of putative regulatory elements. Results The performance of linear and quadratic discriminant analysis (LDA, QDA in identifying NF-κB-responding genes was examined on a dataset of 240 positive and negative examples of NF-κB regulation, using stratified cross-validation with an internal leave-one-out cross-validation (LOOCV loop for automated feature selection and noise reduction. LDA performed slightly better than QDA, achieving 61% sensitivity, 91% specificity and 87% positive predictive value, and allowing the identification of 231, 251 and 580 NF-κB putative target genes in insulin-producing INS-1E cells, primary rat beta-cells and human pancreatic islets, respectively. Predicted NF-κB targets had a significant enrichment in genes regulated by cytokines (IL-1β or IL-1β + IFN-γ and double stranded RNA (dsRNA, as compared to genes not regulated by these NF-κB-dependent stimuli. We increased the confidence of the predictions by selecting only evolutionary stable genes, i.e. genes with homologs predicted as NF-κB targets in rat, mouse, human and chimpanzee. Conclusion The present in silico analysis allowed us to identify novel regulatory targets of NF-κB using a supervised classification method based on

PARP-1 and YY1 are important novel regulators of CXCL12 gene transcription in rat pancreatic beta cells.

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Jelena Marković

Full Text Available Despite significant progress, the molecular mechanisms responsible for pancreatic beta cell depletion and development of diabetes remain poorly defined. At present, there is no preventive measure against diabetes. The positive impact of CXCL12 expression on the pancreatic beta cell prosurvival phenotype initiated this study. Our aim was to provide novel insight into the regulation of rat CXCL12 gene (Cxcl12 transcription. The roles of poly(ADP-ribose polymerase-1 (PARP-1 and transcription factor Yin Yang 1 (YY1 in Cxcl12 transcription were studied by examining their in vitro and in vivo binding affinities for the Cxcl12 promoter in a pancreatic beta cell line by the electrophoretic mobility shift assay and chromatin immunoprecipitation. The regulatory activities of PARP-1 and YY1 were assessed in transfection experiments using a reporter vector with a Cxcl12 promoter sequence driving luciferase gene expression. Experimental evidence for PARP-1 and YY1 revealed their trans-acting potential, wherein PARP-1 displayed an inhibitory, and YY1 a strong activating effect on Cxcl12 transcription. Streptozotocin (STZ-induced general toxicity in pancreatic beta cells was followed by changes in Cxcl12 promoter regulation. PARP-1 binding to the Cxcl12 promoter during basal and in STZ-compromised conditions led us to conclude that PARP-1 regulates constitutive Cxcl12 expression. During the early stage of oxidative stress, YY1 exhibited less affinity toward the Cxcl12 promoter while PARP-1 displayed strong binding. These interactions were accompanied by Cxcl12 downregulation. In the later stages of oxidative stress and intensive pancreatic beta cell injury, YY1 was highly expressed and firmly bound to Cxcl12 promoter in contrast to PARP-1. These interactions resulted in higher Cxcl12 expression. The observed ability of PARP-1 to downregulate, and of YY1 to upregulate Cxcl12 promoter activity anticipates corresponding effects in the natural context where the

Nuclear import of glucokinase in pancreatic beta-cells is mediated by a nuclear localization signal and modulated by SUMOylation.

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Johansson, Bente Berg; Fjeld, Karianne; Solheim, Marie Holm; Shirakawa, Jun; Zhang, Enming; Keindl, Magdalena; Hu, Jiang; Lindqvist, Andreas; Døskeland, Anne; Mellgren, Gunnar; Flatmark, Torgeir; Njølstad, Pål Rasmus; Kulkarni, Rohit N; Wierup, Nils; Aukrust, Ingvild; Bjørkhaug, Lise

2017-10-15

The localization of glucokinase in pancreatic beta-cell nuclei is a controversial issue. Although previous reports suggest such a localization, the mechanism for its import has so far not been identified. Using immunofluorescence, subcellular fractionation and mass spectrometry, we present evidence in support of glucokinase localization in beta-cell nuclei of human and mouse pancreatic sections, as well as in human and mouse isolated islets, and murine MIN6 cells. We have identified a conserved, seven-residue nuclear localization signal ( 30 LKKVMRR 36 ) in the human enzyme. Substituting the residues KK 31,32 and RR 35,36 with AA led to a loss of its nuclear localization in transfected cells. Furthermore, our data indicates that SUMOylation of glucokinase modulates its nuclear import, while high glucose concentrations do not significantly alter the enzyme nuclear/cytosolic ratio. Thus, for the first time, we provide data in support of a nuclear import of glucokinase mediated by a redundant mechanism, involving a nuclear localization signal, and which is modulated by its SUMOylation. These findings add new knowledge to the functional role of glucokinase in the pancreatic beta-cell. Copyright © 2017 Elsevier B.V. All rights reserved.

Islet-selectivity of G-protein coupled receptor ligands evaluated for PET imaging of pancreatic {beta}-cell mass

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Cline, Gary W., E-mail: gary.cline@yale.edu [Yale University School of Medicine (United States); Zhao, Xiaojian [Yale University School of Medicine (United States); Jakowski, Amy B.; Soeller, Walter C.; Treadway, Judith L. [Pfizer Global Research and Development, Pfizer Inc., Groton CT (United States)

2011-09-02

Highlights: {yields} We screened G-protein coupled receptors for imaging pancreatic. {yields} Database mining and immunohistochemistry identified GPCRs enriched in {beta}-cells. {yields} In vitro and in vivo assays were used to determine exocrine vs endocrine specificity. {yields} GPCR candidates for imaging of {beta}-cell mass are Prokineticin-1R, mGluR5, and GLP-1R. -- Abstract: A critical unmet need exists for methods to quantitatively measure endogenous pancreatic {beta}-cell mass (BCM) for the clinical evaluation of therapies to prevent or reverse loss of BCM and diabetes progression. Our objective was to identify G-protein coupled receptors (GPCRs) that are expressed with a high degree of specificity to islet {beta}-cells for receptor-targeted imaging of BCM. GPCRs enriched in pancreatic islets relative to pancreas acinar and hepatic tissue were identified using a database screen. Islet-specific expression was confirmed by human pancreas immunohistochemistry (IHC). In vitro selectivity assessment was determined from the binding and uptake of radiolabeled ligands to the rat insulinoma INS-1 832/13 cell line and isolated rat islets relative to the exocrine pancreas cell-type, PANC-1. Tail-vein injections of radioligands into rats were used to determine favorable image criteria of in vivo biodistribution to the pancreas relative to other internal organs (i.e., liver, spleen, stomach, and lungs). Database and IHC screening identified four candidate receptors for further in vitro and in vivo evaluation for PET imaging of BCM: prokineticin-1 receptor (PK-1R), metabotropic glutamate receptor type-5 (mGluR5), neuropeptide Y-2 receptor (NPY-2R), and glucagon-like peptide 1 receptor (GLP-1R). In vitro specificity ratios gave the following receptor rank order: PK-1R > GLP-1R > NPY-2R > mGluR5. The biodistribution rank order of selectivity to the pancreas was found to be PK-1R > VMAT2 {approx} GLP-1R > mGluR5. Favorable islet selectivity and biodistribution

Development of biomarker specific of pancreatic beta cells (incretin radiolabelled) for image of beta functional mass in diabetic and obese: study in animal model

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Seo, Daniele

2017-01-01

Increased prevalence of obesity worldwide, has become a vast concern, stimulating investigations focusing prevention and therapy of this condition. The association of type 2 diabetes or insulin resistance aggravates the prognosis of obesity. Even patients successfully submitted to bariatric or metabolic surgery, may not be cured of diabetes, as improvement of circulating values of glucose and insulin not necessarily reflects recovery of pancreatic beta cell mass. There is no consensus about how to estimate beta cell mass in vivo. Available tools suffer from low sensitivity and specificity, often being as well cumbersome and expensive. Radiolabeled incretins, such as glucagon-like-peptide 1 (GLP-1) analogs, seem to be promising options for the measurement of beta cell mass in diabetes and insulinoma. The objective of this study was the development of two conjugates of GLP-1 analog, radiolabeled with 99m Technetium, as a noninvasive imaging method for the estimation of pancreatic beta cell mass, in the presence of obesity. Animal models were selected, including hyperlipidic diet-induced obesity, diet restricted obesity, and as controls, alloxan diabetes. Results indicated that both radiotracers achieved over 97% radiochemical yield. The most successful product was 99m Tc-HYNIC-βAla-Exendin-4. Low beta cell mass uptake occurred in diet-induced obesity. Diet-restricted obesity, with substantial shedding of excess body weight, was followed by remarkable decrease of fasting blood glucose, however beta cell mass uptake was only mildly improved. Future studies are recommended in obesity, type 2 diabetes, and dieting, including bariatric and metabolic operations. (author)

Increased androgen levels in rats impair glucose-stimulated insulin secretion through disruption of pancreatic beta cell mitochondrial function.

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Wang, Hongdong; Wang, Xiaping; Zhu, Yunxia; Chen, Fang; Sun, Yujie; Han, Xiao

2015-11-01

Although insulin resistance is recognized to contribute to the reproductive and metabolic phenotypes of polycystic ovary syndrome (PCOS), pancreatic beta cell dysfunction plays an essential role in the progression from PCOS to the development of type 2 diabetes. However, the role of insulin secretory abnormalities in PCOS has received little attention. In addition, the precise changes in beta cells and the underlying mechanisms remain unclear. In this study, we therefore attempted to elucidate potential mechanisms involved in beta cell alterations in a rat model of PCOS. Glucose-induced insulin secretion was measured in islets isolated from DHT-treated and control rats. Oxygen consumption rate (OCR), ATP production, and mitochondrial copy number were assayed to evaluate mitochondrial function. Glucose-stimulated insulin secretion is significantly decreased in islets from DHT-treated rats. On the other hand, significant reductions are observed in the expression levels of several key genes involved in mitochondrial biogenesis and in mitochondrial OCR and ATP production in DHT-treated rat islets. Meanwhile, we found that androgens can directly impair beta cell function by inducing mitochondrial dysfunction in vitro in an androgen receptor dependent manner. For the first time, our study demonstrates that increased androgens in female rats can impair glucose-stimulated insulin secretion partly through disruption of pancreatic beta cell mitochondrial function. This work has significance for hyperandrogenic women with PCOS: excess activation of the androgen receptor by androgens may provoke beta cell dysfunction via mitochondrial dysfunction. Copyright © 2015 Elsevier Ltd. All rights reserved.

Clusters of conserved beta cell marker genes for assessment of beta cell phenotype

DEFF Research Database (Denmark)

Martens, Geert A; Jiang, Lei; Hellemans, Karine H

2011-01-01

The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...... of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture...... microdissected beta cells, monitor adaptations of the beta cell phenotype to fasting, and retrieve possible conserved transcriptional regulators....

Vanadyl Sulfate Treatment Stimulates Proliferation and Regeneration of Beta Cells in Pancreatic Islets

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Samira Missaoui

2014-01-01

Full Text Available We examined the effects of vanadium sulfate (VOSO4 treatment at 5 and 10 mg/kg for 30 days on endocrine pancreas activity and histology in nondiabetic and STZ-induced diabetic rats. In diabetic group, blood glucose levels significantly increased while insulinemia level markedly decreased. At the end of treatment, VOSO4 at a dose of 10 mg/Kg normalized blood glucose level in diabetic group, restored insulinemia, and significantly improved insulin sensitivity. VOSO4 also increased in a dose-dependent manner the number of insulin immunopositive beta cells in pancreatic islets of nondiabetic rats. Furthermore, in the STZ-diabetic group, the decrease in the number of insulin immunopositive beta cells was corrected to reach the control level mainly with the higher dose of vanadium. Therefore, VOSO4 treatment normalized plasma glucose and insulin levels and improved insulin sensitivity in STZ-experimental diabetes and induced beta cells proliferation and/or regeneration in normal or diabetic rats.

Activation of transmembrane bile acid receptor TGR5 stimulates insulin secretion in pancreatic {beta} cells

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Kumar, Divya P.; Rajagopal, Senthilkumar; Mahavadi, Sunila [Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Mirshahi, Faridoddin [Division of Gastroenterology, Hepatology and Nutrition, Department of Internal Medicine, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Grider, John R. [Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Murthy, Karnam S., E-mail: skarnam@vcu.edu [Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Sanyal, Arun J., E-mail: asanyal@mcvh-vcu.edu [Division of Gastroenterology, Hepatology and Nutrition, Department of Internal Medicine, Virginia Commonwealth University School of Medicine, Richmond, VA (United States)

2012-10-26

Highlights: Black-Right-Pointing-Pointer G protein coupled receptor TGR5 is expressed in mouse and human islets. Black-Right-Pointing-Pointer TGR5 is coupled to activation of Gs and Ca{sup 2+} release via cAMP/Epac/PLC-{epsilon} pathway. Black-Right-Pointing-Pointer Activation of TGR5 by bile salts and selective ligands causes insulin secretion. Black-Right-Pointing-Pointer TGR5 could be a potential therapeutic target to treat diabetes. -- Abstract: Bile acids act as signaling molecules and stimulate the G protein coupled receptor, TGR5, in addition to nuclear farnesoid X receptor to regulate lipid, glucose and energy metabolism. Bile acid induced activation of TGR5 in the enteroendocrine cells promotes glucagon like peptide-1 (GLP-1) release, which has insulinotropic effect in the pancreatic {beta} cells. In the present study, we have identified the expression of TGR5 in pancreatic {beta} cell line MIN6 and also in mouse and human pancreatic islets. TGR5 selective ligands, oleanolic acid (OA) and INT-777 selectively activated G{alpha}{sub s} and caused an increase in intracellular cAMP and Ca{sup 2+}. OA and INT-777 also increased phosphoinositide (PI) hydrolysis and the increase was blocked by NF449 (a selective G{alpha}{sub s} inhibitor) or (U73122) (PI hydrolysis inhibitor). OA, INT-777 and lithocholic acid increased insulin release in MIN6 and human islets and the increase was inhibited by treatment with NF449, (U73122) or BAPTA-AM (chelator of calcium), but not with myristoylated PKI (PKA inhibitor), suggesting that the release is dependent on G{sub s}/cAMP/Ca{sup 2+} pathway. 8-pCPT-2 Prime -O-Me-cAMP, a cAMP analog, which activates Epac, but not PKA also stimulated PI hydrolysis. In conclusion, our study demonstrates that the TGR5 expressed in the pancreatic {beta} cells regulates insulin secretion and highlights the importance of ongoing therapeutic strategies targeting TGR5 in the control of glucose homeostasis.

Clusters of conserved beta cell marker genes for assessment of beta cell phenotype

DEFF Research Database (Denmark)

Martens, Geert A; Jiang, Lei; Hellemans, Karine H

2011-01-01

The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...... of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture...

STAT5 activity in pancreatic beta-cells influences the severity of diabetes in animal models of type 1 and 2 diabetes

DEFF Research Database (Denmark)

Jackerott, Malene; Møldrup, Annette; Thams, Peter

2006-01-01

Pancreatic beta-cell growth and survival and insulin production are stimulated by growth hormone and prolactin through activation of the transcription factor signal transducer and activator of transcription (STAT)5. To assess the role of STAT5 activity in beta-cells in vivo, we generated transgen...... and type 2 diabetes....... reduced beta-cell proliferation at 6 months of age. The inhibitory effect of high-fat diet or leptin on insulin secretion was diminished in isolated islets from RIP-DNSTAT5 mice compared with wild-type islets. Upon multiple low-dose streptozotocin treatment, RIP-DNSTAT5 mice exhibited higher plasma...... of glucose tolerance, whereas RIP-CASTAT5 mice were more glucose tolerant and less hyperleptinemic than wild-type mice. Although the pancreatic insulin content and relative beta-cell area were increased in high-fat diet-fed RIP-DNSTAT5 mice compared with wild-type or RIP-CASTAT5 mice, RIP-DNSTAT5 mice showed...

Stearoyl-CoA Desaturase-1 Protects Cells against Lipotoxicity-Mediated Apoptosis in Proximal Tubular Cells

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Tamaki Iwai

2016-11-01

Full Text Available Saturated fatty acid (SFA-related lipotoxicity is a pathogenesis of diabetes-related renal proximal tubular epithelial cell (PTEC damage, closely associated with a progressive decline in renal function. This study was designed to identify a free fatty acid (FFA metabolism-related enzyme that can protect PTECs from SFA-related lipotoxicity. Among several enzymes involved in FFA metabolism, we identified stearoyl-CoA desaturase-1 (SCD1, whose expression level significantly decreased in the kidneys of high-fat diet (HFD-induced diabetic mice, compared with non-diabetic mice. SCD1 is an enzyme that desaturates SFAs, converting them to monounsaturated fatty acids (MUFAs, leading to the formation of neutral lipid droplets. In culture, retrovirus-mediated overexpression of SCD1 or MUFA treatment significantly ameliorated SFA-induced apoptosis in PTECs by enhancing intracellular lipid droplet formation. In contrast, siRNA against SCD1 exacerbated the apoptosis. Both overexpression of SCD1 and MUFA treatment reduced SFA-induced apoptosis via reducing endoplasmic reticulum stress in cultured PTECs. Thus, HFD-induced decrease in renal SCD1 expression may play a pathogenic role in lipotoxicity-induced renal injury, and enhancing SCD1-mediated desaturation of SFA and subsequent formation of neutral lipid droplets may become a promising therapeutic target to reduce SFA-induced lipotoxicity. The present study provides a novel insight into lipotoxicity in the pathogenesis of diabetic nephropathy.

Effects of ethanol on pancreatic beta-cell death: interaction with glucose and fatty acids.

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Dembele, Korami; Nguyen, K Hoa; Hernandez, Tiffany A; Nyomba, B L Grégoire

2009-04-01

Western lifestyle plays an important role in the prevalence of type 2 diabetes by causing insulin resistance and pancreatic beta-cell dysfunction, a prerequisite for the development of diabetes. High fat diet and alcohol are major components of the western diet. The aim of the present study was to investigate the effects of ethanol and fatty acids on beta-cell survival and metabolism. We treated the rat beta-cell line RINm5F with ethanol, a mixture of palmitic and oleic acids, or both. Reactive oxygen species (ROS) were determined by (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate) (CM-H2DCFDA) fluorescence assay, and mitochondrial activity was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction assay and by determining ATP production. Cell viability was assessed with a cell counter and trypan blue exclusion, and the mode of cell death by Hoechst33342 and propidium iodide staining. With both ethanol and fatty acid treatments, MTT reduction and ATP production decreased, whereas ROS production increased. Ethanol treatment had no effect on cell number, whereas fatty acid treatment reduced the cell number. Cell incubation with ethanol, fatty acids, or both increased the number of Hoechst 33342-positive nuclei. However, the majority of nuclei from fatty acid-treated cells were stained with propidium iodide, indicating a loss of plasma membrane integrity. We conclude that both ethanol and fatty acids generate cellular oxidative stress, and affect mitochondrial function in RINm5F beta-cells. However, ethanol causes beta-cell death by apoptosis, whereas fatty acids cause cell death predominantly by necrosis. It is not known whether these results are applicable to human beta-cells.

Involvement of interleukin 1 and interleukin 1 antagonist in pancreatic beta-cell destruction in insulin-dependent diabetes mellitus

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Mandrup-Poulsen, T; Zumsteg, U; Reimers, J

1993-01-01

In this review we propose that the balance between the action of interleukin 1 (IL-1) and its natural antagonist IL-1ra on the level of the insulin-producing pancreatic beta-cell may play a decisive role in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). We argue that IL-1...... potentiated by other cytokines (tumor necrosis factor alpha, interferon gamma) is an important effector molecule involved in both early and late events in the immune-mediated process that leads to beta-cell destruction and IDDM. We also point out that surprisingly high molar excesses of IL-1ra over IL-1...... are necessary to block the action of IL-1 on islet beta-cells compared to islet alpha-cells in vitro and in animals. We suggest that the selectivity of beta-cell destruction in IDDM may be conferred on several levels: (1) homing of beta-cell antigen specific T cells, (2) targeted delivery of cytokines...

Pathway decision-making strategies for generating pancreatic beta-cells: systems biology or hit and miss?

Science.gov (United States)

Jensen, Jan

2007-08-01

Method selection strategies to achieve beta-cell differentiation from human embryonic stem cells are reviewed. Expectations are high for an embryonic stem cell derived cellular replacement method to cure diabetes, and recent observations indicate that mature beta-cells can be derived from human embryonic stem cells. In terms of the translational setting, however, we are not there yet; conversion of embryonic stem cells to fully mature beta-cells is not effective and does not provide an exclusive beta-cell population as the end-product. Studies emphasize that expecting development of a 'magic media bullet' that promotes endocrine fate is rather optimistic; consecutive administration of signaling inducers, carefully provided to mimic normal development, is more likely to succeed. To accomplish this task we need better understanding of the extracellular signaling pathways that drive progressive endodermal fate choices throughout development. Knowledge of cell-intrinsic control of fate assignments in pancreas is growing rapidly. Nevertheless, insufficient information regarding morphogen codes that operate in endoderm and pancreas is hindering development of better, directed differentiation schema of uncommitted embryonic stem cells toward endodermal, pancreatic, and subsequent endocrine specific fates. A systematic approach to defining morphogen codes in developing endoderm and pancreas appears timely and justified.

Antioxidant and regulatory role of mitochondrial uncoupling protein UCP2 in pancreatic beta-cells

Czech Academy of Sciences Publication Activity Database

Ježek, Petr; Olejár, Tomáš; Smolková, Katarína; Ježek, Jan; Dlasková, Andrea; Plecitá-Hlavatá, Lydie; Zelenka, Jaroslav; Špaček, Tomáš; Engstová, Hana; Reguera Pajuelo, David; Jabůrek, Martin

2014-01-01

Roč. 63, Suppl.1 (2014), S73-S91 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GAP302/10/0346; GA ČR(CZ) GAP305/12/1247; GA ČR(CZ) GPP304/10/P204; GA MŠk(CZ) EE2.3.30.0025 Institutional support: RVO:67985823 Keywords : mitochondria * uncoupling protein UCP2 * pancreatic beta-cells * reactive oxygen species * glucose-stimulated insulin secretion Subject RIV: EA - Cell Biology Impact factor: 1.293, year: 2014

EX4 stabilizes and activates Nrf2 via PKCδ, contributing to the prevention of oxidative stress-induced pancreatic beta cell damage

Energy Technology Data Exchange (ETDEWEB)

Kim, Mi-Hwi; Kim, Eung-Hwi [College of Pharmacy, Gachon Institute of Pharmaceutical Science, Gachon University, Yeonsu-ku, Incheon (Korea, Republic of); Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Yeonsu-ku, Incheon (Korea, Republic of); Jung, Hye Seung [Department of Internal Medicine, Seoul National University College of Medicine, Seoul (Korea, Republic of); Yang, Dongki [Department of Physiology, Gachon University College of Medicine, Incheon (Korea, Republic of); Park, Eun-Young, E-mail: parkey@mokpo.ac.kr [College of Pharmacy, Natural Medicine Research Institute, Mokpo National University, Muan-gun, Jeonnam (Korea, Republic of); Jun, Hee-Sook, E-mail: hsjun@gachon.ac.kr [College of Pharmacy, Gachon Institute of Pharmaceutical Science, Gachon University, Yeonsu-ku, Incheon (Korea, Republic of); Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Yeonsu-ku, Incheon (Korea, Republic of); Gachon Medical Research Institute, Gil Hospital, Incheon (Korea, Republic of)

2017-01-15

Oxidative stress in pancreatic beta cells can inhibit insulin secretion and promote apoptotic cell death. Exendin-4 (EX4), a glucagon-like peptide-1 receptor agonist, can suppress beta cell apoptosis, improve beta cell function and protect against oxidative damage. In this study, we investigated the molecular mechanisms for antioxidative effects of EX4 in pancreatic beta cells. INS-1 cells, a rat insulinoma cell line, were pretreated with EX4 and exposed to palmitate or H{sub 2}O{sub 2}. Reactive oxygen species (ROS) production, and glutathione and insulin secretion were measured. The mRNA and protein expression levels of antioxidant genes were examined. The level of nuclear factor erythroid 2-related factor 2 (Nrf2), its binding to antioxidant response element (ARE), and its ubiquination in the presence of EX4 were determined. The Nrf2 signaling pathway was determined using rottlerin (protein kinase [PK]Cδ inhibitor), H89 (PKA inhibitor) and LY294002 (phosphatidylinositide 3-kinase [PI3K] inhibitor). EX4 treatment decreased ROS production, recovered cellular glutathione levels and insulin secretion in the presence of oxidative stress in INS-1 cells. The expression levels of glutamate-cysteine ligase catalytic subunit and heme oxygenase-1 were increased by EX4 treatment. EX4 promoted Nrf2 translocation, ARE binding activity and enhanced stabilization of Nrf2 by inhibition of ubiquitination. Knockdown of Nrf2 abolished the effect of EX4 on increased insulin secretion. Inhibition of PKCδ attenuated Nrf2 translocation and antioxidative gene expression by EX4 treatment. We suggest that EX4 activates and stabilizes Nrf2 through PKCδ activation, contributing to the increase of antioxidant gene expression and consequently improving beta cell function in the presence of oxidative stress. - Highlights: • EX4 protects against oxidative stress-induced pancreatic beta cell dysfunction. • EX4 increases antioxidant gene expression. • Antioxidative effect of EX4 is

EX4 stabilizes and activates Nrf2 via PKCδ, contributing to the prevention of oxidative stress-induced pancreatic beta cell damage

International Nuclear Information System (INIS)

Kim, Mi-Hwi; Kim, Eung-Hwi; Jung, Hye Seung; Yang, Dongki; Park, Eun-Young; Jun, Hee-Sook

2017-01-01

Oxidative stress in pancreatic beta cells can inhibit insulin secretion and promote apoptotic cell death. Exendin-4 (EX4), a glucagon-like peptide-1 receptor agonist, can suppress beta cell apoptosis, improve beta cell function and protect against oxidative damage. In this study, we investigated the molecular mechanisms for antioxidative effects of EX4 in pancreatic beta cells. INS-1 cells, a rat insulinoma cell line, were pretreated with EX4 and exposed to palmitate or H 2 O 2 . Reactive oxygen species (ROS) production, and glutathione and insulin secretion were measured. The mRNA and protein expression levels of antioxidant genes were examined. The level of nuclear factor erythroid 2-related factor 2 (Nrf2), its binding to antioxidant response element (ARE), and its ubiquination in the presence of EX4 were determined. The Nrf2 signaling pathway was determined using rottlerin (protein kinase [PK]Cδ inhibitor), H89 (PKA inhibitor) and LY294002 (phosphatidylinositide 3-kinase [PI3K] inhibitor). EX4 treatment decreased ROS production, recovered cellular glutathione levels and insulin secretion in the presence of oxidative stress in INS-1 cells. The expression levels of glutamate-cysteine ligase catalytic subunit and heme oxygenase-1 were increased by EX4 treatment. EX4 promoted Nrf2 translocation, ARE binding activity and enhanced stabilization of Nrf2 by inhibition of ubiquitination. Knockdown of Nrf2 abolished the effect of EX4 on increased insulin secretion. Inhibition of PKCδ attenuated Nrf2 translocation and antioxidative gene expression by EX4 treatment. We suggest that EX4 activates and stabilizes Nrf2 through PKCδ activation, contributing to the increase of antioxidant gene expression and consequently improving beta cell function in the presence of oxidative stress. - Highlights: • EX4 protects against oxidative stress-induced pancreatic beta cell dysfunction. • EX4 increases antioxidant gene expression. • Antioxidative effect of EX4 is mediated by

Inter-domain tagging implicates caveolin-1 in insulin receptor trafficking and Erk signaling bias in pancreatic beta-cells

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Tobias Boothe

2016-05-01

Full Text Available Objective: The role and mechanisms of insulin receptor internalization remain incompletely understood. Previous trafficking studies of insulin receptors involved fluorescent protein tagging at their termini, manipulations that may be expected to result in dysfunctional receptors. Our objective was to determine the trafficking route and molecular mechanisms of functional tagged insulin receptors and endogenous insulin receptors in pancreatic beta-cells. Methods: We generated functional insulin receptors tagged with pH-resistant fluorescent proteins between domains. Confocal, TIRF and STED imaging revealed a trafficking pattern of inter-domain tagged insulin receptors and endogenous insulin receptors detected with antibodies. Results: Surprisingly, interdomain-tagged and endogenous insulin receptors in beta-cells bypassed classical Rab5a- or Rab7-mediated endocytic routes. Instead, we found that removal of insulin receptors from the plasma membrane involved tyrosine-phosphorylated caveolin-1, prior to trafficking within flotillin-1-positive structures to lysosomes. Multiple methods of inhibiting caveolin-1 significantly reduced Erk activation in vitro or in vivo, while leaving Akt signaling mostly intact. Conclusions: We conclude that phosphorylated caveolin-1 plays a role in insulin receptor internalization towards lysosomes through flotillin-1-positive structures and that caveolin-1 helps bias physiological beta-cell insulin signaling towards Erk activation. Author Video: Author Video Watch what authors say about their articles Keywords: Insulin receptor internalization, Insulin resistance, Pancreatic islet beta-cells, Autocrine insulin signaling

Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic beta cells.

Directory of Open Access Journals (Sweden)

Laura Bordone

2006-02-01

Full Text Available Sir2 and insulin/IGF-1 are the major pathways that impinge upon aging in lower organisms. In Caenorhabditis elegans a possible genetic link between Sir2 and the insulin/IGF-1 pathway has been reported. Here we investigate such a link in mammals. We show that Sirt1 positively regulates insulin secretion in pancreatic beta cells. Sirt1 represses the uncoupling protein (UCP gene UCP2 by binding directly to the UCP2 promoter. In beta cell lines in which Sirt1 is reduced by SiRNA, UCP2 levels are elevated and insulin secretion is blunted. The up-regulation of UCP2 is associated with a failure of cells to increase ATP levels after glucose stimulation. Knockdown of UCP2 restores the ability to secrete insulin in cells with reduced Sirt1, showing that UCP2 causes the defect in glucose-stimulated insulin secretion. Food deprivation induces UCP2 in mouse pancreas, which may occur via a reduction in NAD (a derivative of niacin levels in the pancreas and down-regulation of Sirt1. Sirt1 knockout mice display constitutively high UCP2 expression. Our findings show that Sirt1 regulates UCP2 in beta cells to affect insulin secretion.

The Role of Oxidative Stress and Hypoxia in Pancreatic Beta-Cell Dysfunction in Diabetes Mellitus.

Science.gov (United States)

Gerber, Philipp A; Rutter, Guy A

2017-04-01

Metabolic syndrome is a frequent precursor of type 2 diabetes mellitus (T2D), a disease that currently affects ∼8% of the adult population worldwide. Pancreatic beta-cell dysfunction and loss are central to the disease process, although understanding of the underlying molecular mechanisms is still fragmentary. Recent Advances: Oversupply of nutrients, including glucose and fatty acids, and the subsequent overstimulation of beta cells, are believed to be an important contributor to insulin secretory failure in T2D. Hypoxia has also recently been implicated in beta-cell damage. Accumulating evidence points to a role for oxidative stress in both processes. Although the production of reactive oxygen species (ROS) results from enhanced mitochondrial respiration during stimulation with glucose and other fuels, the expression of antioxidant defense genes is unusually low (or disallowed) in beta cells. Not all subjects with metabolic syndrome and hyperglycemia go on to develop full-blown diabetes, implying an important role in disease risk for gene-environment interactions. Possession of common risk alleles at the SLC30A8 locus, encoding the beta-cell granule zinc transporter ZnT8, may affect cytosolic Zn 2+ concentrations and thus susceptibility to hypoxia and oxidative stress. Loss of normal beta-cell function, rather than total mass, is increasingly considered to be the major driver for impaired insulin secretion in diabetes. Better understanding of the role of oxidative changes, its modulation by genes involved in disease risk, and effects on beta-cell identity may facilitate the development of new therapeutic strategies to this disease. Antioxid. Redox Signal. 26, 501-518.

Islet Cells Serve as Cells of Origin of Pancreatic Gastrin-Positive Endocrine Tumors.

Science.gov (United States)

Bonnavion, Rémy; Teinturier, Romain; Jaafar, Rami; Ripoche, Doriane; Leteurtre, Emmanuelle; Chen, Yuan-Jia; Rehfeld, Jens F; Lepinasse, Florian; Hervieu, Valérie; Pattou, François; Vantyghem, Marie-Christine; Scoazec, Jean-Yves; Bertolino, Philippe; Zhang, Chang Xian

2015-10-01

The cells of origin of pancreatic gastrinomas remain an enigma, since no gastrin-expressing cells are found in the normal adult pancreas. It was proposed that the cellular origin of pancreatic gastrinomas may come from either the pancreatic cells themselves or gastrin-expressing cells which have migrated from the duodenum. In the current study, we further characterized previously described transient pancreatic gastrin-expressing cells using cell lineage tracing in a pan-pancreatic progenitor and a pancreatic endocrine progenitor model. We provide evidence showing that pancreatic gastrin-expressing cells, found from embryonic day 12.5 until postnatal day 7, are derived from pancreatic Ptf1a(+) and neurogenin 3-expressing (Ngn3(+)) progenitors. Importantly, the majority of them coexpress glucagon, with 4% coexpressing insulin, indicating that they are a temporary subpopulation of both alpha and beta cells. Interestingly, Men1 disruption in both Ngn3 progenitors and beta and alpha cells resulted in the development of pancreatic gastrin-expressing tumors, suggesting that the latter developed from islet cells. Finally, we detected gastrin expression using three human cohorts with pancreatic endocrine tumors (pNETs) that have not been diagnosed as gastrinomas (in 9/34 pNETs from 6/14 patients with multiple endocrine neoplasia type 1, in 5/35 sporadic nonfunctioning pNETs, and in 2/20 sporadic insulinomas), consistent with observations made in mouse models. Our work provides insight into the histogenesis of pancreatic gastrin-expressing tumors. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Adult Human Pancreatic Islet Beta-Cells Display Limited Turnover and Long Lifespan as Determined by In-Vivo Thymidine Analog Incorporation and Radiocarbon Dating

Energy Technology Data Exchange (ETDEWEB)

Perl, S; Kushner, J A; Buchholz, B A; Meeker, A K; Stein, G M; Hsieh, M; Kirby, M; Pechhold, S; Liu, E H; Harlan, D M; Tisdale, J F

2010-03-15

Diabetes mellitus results from an absolute or relative deficiency of insulin producing pancreatic beta-cells. The adult human beta-cell's turnover rate remains unknown. We employed novel techniques to examine adult human islet beta-cell turnover and longevity in vivo. Subjects enrolled in NIH clinical trials received thymidine analogues [iododeoxyuridine (IdU) or bromodeoxyuridine (BrdU)] 8-days to 4-years prior to death. Archival autopsy samples from ten patients (aged 17-74 years) were employed to assess beta-cell turnover by scoring nuclear analog labeling within insulin staining cells. Human adult beta-cell longevity was determined by estimating the cells genomic DNA integration of atmospheric carbon-14 ({sup 14}C). DNA was purified from pancreatic islets isolated from cadaveric donors; whole islet prep DNA was obtained from a 15 year old donor, and purified beta-cell DNA was obtained from two donors (age 48 and 80 years). {sup 14}C levels were then determined using accelerator mass spectrometry (AMS). Cellular 'birth date' was determined by comparing the subject's DNA {sup 14}C content relative to a well-established {sup 14}C atmospheric prevalence curve. In the two subjects less than age 20 years, 1-2% of the beta-cell nuclei co-stained for BrdU/IdU. No beta-cell nuclei co-stained in the eight patients more than 30 years old. Consistent with the BrdU/IdU turnover data, beta-cell DNA {sup 14}C content indicated the cells 'birth date' occurred within the subject's first 30 years of life. Under typical circumstances, adult human beta-cells and their cellular precursors are established by young adulthood.

Pancreatic beta cell function increases in a linear dose-response manner following exercise training in adults with prediabetes

DEFF Research Database (Denmark)

Malin, Steven K; Solomon, Thomas; Blaszczak, Alecia

2013-01-01

While some studies suggest that a linear dose-response relationship exists between exercise and insulin sensitivity, the exercise dose required to enhance pancreatic beta-cell function is unknown. Thirty-five older, obese adults with prediabetes underwent a progressive 12-week supervised exercise...

Glucocorticoids Inhibit Basal and Hormone-Induced Serotonin Synthesis in Pancreatic Beta Cells.

Directory of Open Access Journals (Sweden)

Moina Hasni Ebou

Full Text Available Diabetes is a major complication of chronic Glucocorticoids (GCs treatment. GCs induce insulin resistance and also inhibit insulin secretion from pancreatic beta cells. Yet, a full understanding of this negative regulation remains to be deciphered. In the present study, we investigated whether GCs could inhibit serotonin synthesis in beta cell since this neurotransmitter has been shown to be involved in the regulation of insulin secretion. To this aim, serotonin synthesis was evaluated in vitro after treatment with GCs of either islets from CD1 mice or MIN6 cells, a beta-cell line. We also explored the effect of GCs on the stimulation of serotonin synthesis by several hormones such as prolactin and GLP 1. We finally studied this regulation in islet in two in vivo models: mice treated with GCs and with liraglutide, a GLP1 analog, and mice deleted for the glucocorticoid receptor in the pancreas. We showed in isolated islets and MIN6 cells that GCs decreased expression and activity of the two key enzymes of serotonin synthesis, Tryptophan Hydroxylase 1 (Tph1 and 2 (Tph2, leading to reduced serotonin contents. GCs also blocked the induction of serotonin synthesis by prolactin or by a previously unknown serotonin activator, the GLP-1 analog exendin-4. In vivo, activation of the Glucagon-like-Peptide-1 receptor with liraglutide during 4 weeks increased islet serotonin contents and GCs treatment prevented this increase. Finally, islets from mice deleted for the GR in the pancreas displayed an increased expression of Tph1 and Tph2 and a strong increased serotonin content per islet. In conclusion, our results demonstrate an original inhibition of serotonin synthesis by GCs, both in basal condition and after stimulation by prolactin or activators of the GLP-1 receptor. This regulation may contribute to the deleterious effects of GCs on beta cells.

Concerted action of p62 and Nrf2 protects cells from palmitic acid-induced lipotoxicity

Energy Technology Data Exchange (ETDEWEB)

Park, Jeong Su [Severance Biomedical Science Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Kang, Dong Hoon [Department of Life Science and Ewha Research Center for Systems Biology (Korea, Republic of); The Research Center for Cell Homeostasis, Ewha Womans University, Seoul 127-750 (Korea, Republic of); Lee, Da Hyun [Severance Biomedical Science Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Bae, Soo Han, E-mail: soohanbae@yuhs.ac [Severance Biomedical Science Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of)

2015-10-09

Nonalcoholic fatty liver disease (NAFLD), frequently associated with obesity and diabetes mellitus, is caused by the accumulation of excess fatty acids within liver cells. Palmitic acid (PA), a common saturated fatty acid found in mammals, induces the generation of reactive oxygen species (ROS) and elicits apoptotic cell death, known as lipotoxicity. However, protective mechanisms against PA-induced lipotoxicity have not been elucidated. In this study, we aimed to clarify the role of p62, an adapter protein in the autophagic process, as well as the nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway, in protecting cells from PA-induced lipotoxicity. The Nrf2-Keap1 pathway is essential for the protection of cells from oxidative stress. p62 enhances its binding to Keap1 and leads to Nrf2 activation. Here, we show that PA potentiates Keap1 degradation and thereby activates the transcription of Nrf2 target genes partially through autophagy. Furthermore, this PA-mediated Keap1 degradation depends on p62. Correspondingly, a lack of p62 attenuates the PA-mediated Nrf2 activation and increases the susceptibility of cells to oxidative stress. These results indicate that p62 plays an important role in protecting cells against lipotoxicity through Keap1 degradation-mediated Nrf2 activation. - Highlights: • PA induces Keap1 downregulation and activates Nrf2 target gene transcription. • PA-induced Keap1 degradation is partly mediated by the autophagic pathway. • PA-induced Keap1 degradation depends on p62. • Ablation of p62 exacerbates PA-mediated apoptotic cell death.

Concerted action of p62 and Nrf2 protects cells from palmitic acid-induced lipotoxicity

International Nuclear Information System (INIS)

Park, Jeong Su; Kang, Dong Hoon; Lee, Da Hyun; Bae, Soo Han

2015-01-01

Nonalcoholic fatty liver disease (NAFLD), frequently associated with obesity and diabetes mellitus, is caused by the accumulation of excess fatty acids within liver cells. Palmitic acid (PA), a common saturated fatty acid found in mammals, induces the generation of reactive oxygen species (ROS) and elicits apoptotic cell death, known as lipotoxicity. However, protective mechanisms against PA-induced lipotoxicity have not been elucidated. In this study, we aimed to clarify the role of p62, an adapter protein in the autophagic process, as well as the nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway, in protecting cells from PA-induced lipotoxicity. The Nrf2-Keap1 pathway is essential for the protection of cells from oxidative stress. p62 enhances its binding to Keap1 and leads to Nrf2 activation. Here, we show that PA potentiates Keap1 degradation and thereby activates the transcription of Nrf2 target genes partially through autophagy. Furthermore, this PA-mediated Keap1 degradation depends on p62. Correspondingly, a lack of p62 attenuates the PA-mediated Nrf2 activation and increases the susceptibility of cells to oxidative stress. These results indicate that p62 plays an important role in protecting cells against lipotoxicity through Keap1 degradation-mediated Nrf2 activation. - Highlights: • PA induces Keap1 downregulation and activates Nrf2 target gene transcription. • PA-induced Keap1 degradation is partly mediated by the autophagic pathway. • PA-induced Keap1 degradation depends on p62. • Ablation of p62 exacerbates PA-mediated apoptotic cell death.

Early peroxisome proliferator-activated receptor gamma regulated genes involved in expansion of pancreatic beta cell mass

Directory of Open Access Journals (Sweden)

Vivas Yurena

2011-12-01

Full Text Available Abstract Background The progression towards type 2 diabetes depends on the allostatic response of pancreatic beta cells to synthesise and secrete enough insulin to compensate for insulin resistance. The endocrine pancreas is a plastic tissue able to expand or regress in response to the requirements imposed by physiological and pathophysiological states associated to insulin resistance such as pregnancy, obesity or ageing, but the mechanisms mediating beta cell mass expansion in these scenarios are not well defined. We have recently shown that ob/ob mice with genetic ablation of PPARγ2, a mouse model known as the POKO mouse failed to expand its beta cell mass. This phenotype contrasted with the appropriate expansion of the beta cell mass observed in their obese littermate ob/ob mice. Thus, comparison of these models islets particularly at early ages could provide some new insights on early PPARγ dependent transcriptional responses involved in the process of beta cell mass expansion Results Here we have investigated PPARγ dependent transcriptional responses occurring during the early stages of beta cell adaptation to insulin resistance in wild type, ob/ob, PPARγ2 KO and POKO mice. We have identified genes known to regulate both the rate of proliferation and the survival signals of beta cells. Moreover we have also identified new pathways induced in ob/ob islets that remained unchanged in POKO islets, suggesting an important role for PPARγ in maintenance/activation of mechanisms essential for the continued function of the beta cell. Conclusions Our data suggest that the expansion of beta cell mass observed in ob/ob islets is associated with the activation of an immune response that fails to occur in POKO islets. We have also indentified other PPARγ dependent differentially regulated pathways including cholesterol biosynthesis, apoptosis through TGF-β signaling and decreased oxidative phosphorylation.

Effects of methyl mercury exposure on pancreatic beta cell development and function.

Science.gov (United States)

Schumacher, Lauren; Abbott, Louise C

2017-01-01

Methyl mercury is an environmental contaminant of worldwide concern. Since the discovery of methyl mercury exposure due to eating contaminated fish as the underlying cause of the Minamata disaster, the scientific community has known about the sensitivity of the developing central nervous system to mercury toxicity. Warnings are given to pregnant women and young children to limit consumption of foods containing methyl mercury to protect the embryonic, fetal and postnatally developing central nervous system. However, evidence also suggests that exposure to methyl mercury or various forms of inorganic mercury may also affect development and function of other organs. Numerous reports indicate a worldwide increase in diabetes, particularly type 2 diabetes. Quite recently, methyl mercury has been shown to have adverse effects on pancreatic beta (β) cell development and function, resulting in insulin resistance and hyperglycemia and may even lead to the development of diabetes. This review discusses possible mechanisms by which methyl mercury exposure may adversely affect pancreatic β cell development and function, and the role that methyl mercury exposure may have in the reported worldwide increase in diabetes, particularly type 2 diabetes. While additional information is needed regarding associations between mercury exposure and specific mechanisms of the pathogenesis of diabetes in the human population, methyl mercury's adverse effects on the body's natural sources of antioxidants suggest that one possible therapeutic strategy could involve supplementation with antioxidants. Thus, it is important that additional investigation be undertaken into the role of methyl mercury exposure and reduced pancreatic β cell function. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Calcium Co-regulates Oxidative Metabolism and ATP Synthase-dependent Respiration in Pancreatic Beta Cells

Science.gov (United States)

De Marchi, Umberto; Thevenet, Jonathan; Hermant, Aurelie; Dioum, Elhadji; Wiederkehr, Andreas

2014-01-01

Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)+ ratio. PMID:24554722

Ins1 Cre knock-in mice for beta cell-specific gene recombination

OpenAIRE

Thorens Bernard; Tarussio David; Maestro Miguel Angel; Maestro Miguel Angel; Rovira Meritxell; Rovira Meritxell; Heikkilä Eija; Ferrer Jorge; Ferrer Jorge; Ferrer Jorge

2013-01-01

Aims/hypothesis Pancreatic beta cells play a central role in the control of glucose homeostasis by secreting insulin to stimulate glucose uptake by peripheral tissues. Understanding the molecular mechanisms that control beta cell function and plasticity has critical implications for the pathophysiology and therapy of major forms of diabetes. Selective gene inactivation in pancreatic beta cells, using the Cre-lox system, is a powerful approach to assess the role of particular genes in beta cel...

Glucagon-like peptide-1 counteracts the detrimental effects of Advanced Glycation End-Products in the pancreatic beta cell line HIT-T 15

International Nuclear Information System (INIS)

Puddu, A.; Storace, D.; Durante, A.; Odetti, P.; Viviani, G.L.

2010-01-01

Research highlights: → GLP-1 prevents AGEs-induced cell death. → GLP-1 prevents AGEs-induced oxidative stress. → GLP-1 ameliorated AGEs-induced cell dysfunction. → GLP-1 attenuates AGEs-induced RAGE increment. → GLP-1 counteracts AGEs-induced pancreatic cell death and dysfunction. -- Abstract: Advanced Glycation End-Products (AGEs), a group of compounds resulting from the non-enzymatic reaction of reducing sugars with the free amino group of proteins, are implicated in diabetic complications. We previously demonstrated that exposure of the pancreatic islet cell line HIT-T 15 to high concentrations of AGEs significantly decreases cell proliferation and insulin secretion, and affects transcription factors regulating insulin gene transcription. The glucagon-like peptide-1 (GLP-1) is an incretin hormone that increases proinsulin biosynthesis, stimulates insulin secretion, and improves pancreatic beta-cell viability. The aim of this work was to investigate the effects of GLP-1 on the function and viability of HIT-T 15 cells cultured with AGEs. HIT-T 15 cells were cultured for 5 days in presence of AGEs alone, or supplemented with 10 nmol/l GLP-1. Cell viability, insulin secretion, redox balance, and expression of the AGEs receptor (RAGE) were then determined. The results showed that GLP-1 protected beta cell against AGEs-induced cell death preventing both apoptosis and necrosis. Moreover, addition of GLP-1 to the AGEs culture medium restored the redox balance, improved the responsiveness to glucose, and attenuated AGEs-induced RAGE expression. These findings provide evidence that GLP-1 protects beta cells from the dangerous effects of AGEs.

What are the potential benefits of clinical beta-cell imaging in diabetes mellitus?

Science.gov (United States)

Göke, Burkhard

2010-05-01

Previously, studies of the endocrine pancreatic beta-cell were mainly performed ex vivo by morphological means. This data supported the analysis of pathophysiological changes in the pancreatic islet during insults such as diabetes mellitus. Metabolic testing of the pancreatic islet by assaying hormone parameters such als plasma insulin or C-peptide combined with more or less sophisticated calculations allowed conclusions about states of insulin resistance or secretory failure. It also allowed certain correlations of endocrine function with beta-cell mass. Today, with firmer pathophysiological concepts about beta-cell failure, modern protocols of islet transplantation, and drugs on the market coming with promises of preservation or even expansion of beta-cell mass in diabetes mellitus it has become very attractive to search for tools measuring beta-cell mass, if possible even repeatingly in the same organism in vivo. From a clinical point of view, the potential of pancreatic beta-cell mass imaging technologies is looked upon with high expectations. Methodologically, the decisive question is whether it is likely that future beta-cell imaging will provide significant advantages over the metabolic methods already in hand. With new in vivo tools, studies of beta-cell mass and function may offer even new approaches stratifying patients to anti-diabetic therapies.

Role of transcription factor KLF11 and its diabetes-associated gene variants in pancreatic beta cell function

Science.gov (United States)

Neve, Bernadette; Fernandez-Zapico, Martin E.; Ashkenazi-Katalan, Vered; Dina, Christian; Hamid, Yasmin H.; Joly, Erik; Vaillant, Emmanuel; Benmezroua, Yamina; Durand, Emmanuelle; Bakaher, Nicolas; Delannoy, Valerie; Vaxillaire, Martine; Cook, Tiffany; Dallinga-Thie, Geesje M.; Jansen, Hans; Charles, Marie-Aline; Clément, Karine; Galan, Pilar; Hercberg, Serge; Helbecque, Nicole; Charpentier, Guillaume; Prentki, Marc; Hansen, Torben; Pedersen, Oluf; Urrutia, Raul; Melloul, Danielle; Froguel, Philippe

2005-01-01

KLF11 (TIEG2) is a pancreas-enriched transcription factor that has elicited significant attention because of its role as negative regulator of exocrine cell growth in vitro and in vivo. However, its functional role in the endocrine pancreas remains to be established. Here, we report, for the first time, to our knowledge, the characterization of KLF11 as a glucose-inducible regulator of the insulin gene. A combination of random oligonucleotide binding, EMSA, luciferase reporter, and chromatin immunoprecipitation assays shows that KLF11 binds to the insulin promoter and regulates its activity in beta cells. Genetic analysis of the KLF11 gene revealed two rare variants (Ala347Ser and Thr220Met) that segregate with diabetes in families with early-onset type 2 diabetes, and significantly impair its transcriptional activity. In addition, analysis of 1,696 type 2 diabetes mellitus and 1,776 normoglycemic subjects show a frequent polymorphic Gln62Arg variant that significantly associates with type 2 diabetes mellitus in North European populations (OR = 1.29, P = 0.00033). Moreover, this variant alters the corepressor mSin3A-binding activity of KLF11, impairs the activation of the insulin promoter and shows lower levels of insulin expression in pancreatic beta cells. In addition, subjects carrying the Gln62Arg allele show decreased plasma insulin after an oral glucose challenge. Interestingly, all three nonsynonymous KLF11 variants show increased repression of the catalase 1 promoter, suggesting a role in free radical clearance that may render beta cells more sensitive to oxidative stress. Thus, both functional and genetic analyses reveal that KLF11 plays a role in the regulation of pancreatic beta cell physiology, and its variants may contribute to the development of diabetes. PMID:15774581

Cocoa Phenolic Extract Protects Pancreatic Beta Cells against Oxidative Stress

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Laura Bravo

2013-07-01

Full Text Available Diabetes mellitus is associated with reductions in glutathione, supporting the critical role of oxidative stress in its pathogenesis. Antioxidant food components such as flavonoids have a protective role against oxidative stress-induced degenerative and age-related diseases. Flavonoids constitute an important part of the human diet; they can be found in most plant foods, including green tea, grapes or cocoa and possess multiple biological activities. This study investigates the chemo-protective effect of a cocoa phenolic extract (CPE containing mainly flavonoids against oxidative stress induced by tert-butylhydroperoxide (t-BOOH on Ins-1E pancreatic beta cells. Cell viability and oxidative status were evaluated. Ins-1E cells treatment with 5–20 μg/mL CPE for 20 h evoked no cell damage and did not alter ROS production. Addition of 50 μM t-BOOH for 2 h increased ROS and carbonyl groups content and decreased reduced glutathione level. Pre-treatment of cells with CPE significantly prevented the t-BOOH-induced ROS and carbonyl groups and returned antioxidant defences to adequate levels. Thus, Ins-1E cells treated with CPE showed a remarkable recovery of cell viability damaged by t-BOOH, indicating that integrity of surviving machineries in the CPE-treated cells was notably protected against the oxidative insult.

Stevioside improves pancreatic beta-cell function during glucotoxicity via regulation of acetyl-CoA carboxylase.

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Chen, Jianguo; Jeppesen, Per Bendix; Nordentoft, Iver; Hermansen, Kjeld

2007-06-01

Chronic hyperglycemia is detrimental to pancreatic beta-cells, causing impaired insulin secretion and beta-cell turnover. The characteristic secretory defects are increased basal insulin secretion (BIS) and a selective loss of glucose-stimulated insulin secretion (GSIS). Several recent studies support the view that the acetyl-CoA carboxylase (ACC) plays a pivotal role for GSIS. We have shown that stevioside (SVS) enhances insulin secretion and ACC gene expression. Whether glucotoxicity influences ACC and whether this action can be counteracted by SVS are not known. To investigate this, we exposed isolated mouse islets as well as clonal INS-1E beta-cells for 48 h to 27 or 16.7 mM glucose, respectively. We found that 48-h exposure to high glucose impairs GSIS from mouse islets and INS-1E cells, an effect that is partly counteracted by SVS. The ACC dephosphorylation inhibitor okadaic acid (OKA, 10(-8) M), and 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR, 10(-4) M), an activator of 5'-AMP protein kinase that phosphorylates ACC, eliminated the beneficial effect of SVS. 5-Tetrade-cyloxy-2-furancarboxylic acid (TOFA), the specific ACC inhibitor, blocked the effect of SVS as well. During glucotoxity, ACC gene expression, ACC protein, and phosphorylated ACC protein were increased in INS-1E beta-cells. SVS pretreatment further increased ACC gene expression with strikingly elevated ACC activity and increased glucose uptake accompanied by enhanced GSIS. Our studies show that glucose is a potent stimulator of ACC and that SVS to some extent counteracts glucotoxicity via increased ACC activity. SVS possesses the potential to alleviate negative effects of glucotoxicity in beta-cells via a unique mechanism of action.

Lipidomic data on lipid droplet triglyceride remodelling associated with protection of breast cancer cells from lipotoxic stress.

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Jarc, Eva; Eichmann, Thomas O; Zimmermann, Robert; Petan, Toni

2018-06-01

The data presented here is related to the research article entitled "Lipid droplets induced by secreted phospholipase A 2 and unsaturated fatty acids protect breast cancer cells from nutrient and lipotoxic stress" by E. Jarc et al., Biochim. Biophys. Acta 1863 (2018) 247-265. Elevated uptake of unsaturated fatty acids and lipid droplet accumulation are characteristic of aggressive cancer cells and have been associated with the cellular stress response. The present study provides lipidomic data on the triacylglycerol (TAG) and phosphatidylcholine (PC) composition of MDA-MB-231 breast cancer cells exposed to docosahexaenoic acid (DHA; 22:6, ω-3). Datasets provide information on the changes in lipid composition induced by depletion of adipose triglyceride lipase (ATGL) and by exogenous addition of secreted phospholipase A 2 (sPLA 2 ) in DHA-treated cells. The presented alterations in lipid composition, mediated by targeting lipid droplet biogenesis and lipolysis, are associated with protection from lipotoxicity and allow further investigation into the role of lipid droplets in the resistance of cancer cells to lipotoxic stress.

H2O2-Activated Mitochondrial Phospholipase iPLA2 gamma Prevents Lipotoxic Oxidative Stress in Synergy with UCP2, Amplifies Signaling via G-Protein-Coupled Receptor GPR40, and Regulates Insulin Secretion in Pancreatic beta-Cells

Czech Academy of Sciences Publication Activity Database

Ježek, Jan; Dlasková, Andrea; Zelenka, Jaroslav; Jabůrek, Martin; Ježek, Petr

2015-01-01

Roč. 23, č. 12 (2015), s. 958-972 ISSN 1523-0864 R&D Projects: GA ČR(CZ) GPP303/11/P320; GA ČR(CZ) GA13-02033S; GA ČR(CZ) GA13-06666S; GA ČR GA15-02051S Institutional support: RVO:67985823 Keywords : mitochondrial phospholipase iPLA2 gamma * uncoupling protein UCP2 * G-protein coupled receptor - 40 * glucose-stimulated insulin secretion * pancreatic beta cells Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 7.093, year: 2015

Role of pancreatic fat in the outcomes of pancreatitis.

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Acharya, Chathur; Navina, Sarah; Singh, Vijay P

2014-01-01

The role of obesity in relation to various disease processes is being increasingly studied, with reports over the last several years increasingly mentioning its association with worse outcomes in acute disease. Obesity has also gained recognition as a risk factor for severe acute pancreatitis (SAP).The mortality in SAP may be as high as 30% and is usually attributable to multi system organ failure (MSOF) earlier in the disease, and complications of necrotizing pancreatitis later [9-11]. To date there is no specific treatment for acute pancreatitis (AP) and the management is largely expectant and supportive. Obesity in general has also been associated with poor outcomes in sepsis and other pathological states including trauma and burns. With the role of unsaturated fatty acids (UFA) as propagators in SAP having recently come to light and with the recognition of acute lipotoxicity, there is now an opportunity to explore different strategies to reduce the mortality and morbidity in SAP and potentially other disease states associated with such a pathophysiology. In this review we will discuss the role of fat and implications of the consequent acute lipotoxicity on the outcomes of acute pancreatitis in lean and obese states and during acute on chronic pancreatitis. Copyright © 2014 IAP and EPC. Published by Elsevier B.V. All rights reserved.

PED/PEA-15 inhibits hydrogen peroxide-induced apoptosis in Ins-1E pancreatic beta-cells via PLD-1.

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Francesca Fiory

Full Text Available The small scaffold protein PED/PEA-15 is involved in several different physiologic and pathologic processes, such as cell proliferation and survival, diabetes and cancer. PED/PEA-15 exerts an anti-apoptotic function due to its ability to interfere with both extrinsic and intrinsic apoptotic pathways in different cell types. Recent evidence shows that mice overexpressing PED/PEA-15 present larger pancreatic islets and increased beta-cells mass. In the present work we investigated PED/PEA-15 role in hydrogen peroxide-induced apoptosis in Ins-1E beta-cells. In pancreatic islets isolated from Tg(PED/PEA-15 mice hydrogen peroxide-induced DNA fragmentation was lower compared to WT islets. TUNEL analysis showed that PED/PEA-15 overexpression increases the viability of Ins-1E beta-cells and enhances their resistance to apoptosis induced by hydrogen peroxide exposure. The activity of caspase-3 and the cleavage of PARP-1 were markedly reduced in Ins-1E cells overexpressing PED/PEA-15 (Ins-1E(PED/PEA-15. In parallel, we observed a decrease of the mRNA levels of pro-apoptotic genes Bcl-xS and Bad. In contrast, the expression of the anti-apoptotic gene Bcl-xL was enhanced. Accordingly, DNA fragmentation was higher in control cells compared to Ins-1E(PED/PEA-15 cells. Interestingly, the preincubation with propranolol, an inhibitor of the pathway of PLD-1, a known interactor of PED/PEA-15, responsible for its deleterious effects on glucose tolerance, abolishes the antiapoptotic effects of PED/PEA-15 overexpression in Ins-1E beta-cells. The same results have been obtained by inhibiting PED/PEA-15 interaction with PLD-1 in Ins-1E(PED/PEA-15. These results show that PED/PEA-15 overexpression is sufficient to block hydrogen peroxide-induced apoptosis in Ins-1E cells through a PLD-1 mediated mechanism.

Characterization of the distal promoter of the human pyruvate carboxylase gene in pancreatic beta cells.

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Ansaya Thonpho

Full Text Available Pyruvate carboxylase (PC is an enzyme that plays a crucial role in many biosynthetic pathways in various tissues including glucose-stimulated insulin secretion. In the present study, we identify promoter usage of the human PC gene in pancreatic beta cells. The data show that in the human, two alternative promoters, proximal and distal, are responsible for the production of multiple mRNA isoforms as in the rat and mouse. RT-PCR analysis performed with cDNA prepared from human liver and islets showed that the distal promoter, but not the proximal promoter, of the human PC gene is active in pancreatic beta cells. A 1108 bp fragment of the human PC distal promoter was cloned and analyzed. It contains no TATA box but possesses two CCAAT boxes, and other putative transcription factor binding sites, similar to those of the distal promoter of rat PC gene. To localize the positive regulatory region in the human PC distal promoter, 5'-truncated and the 25-bp and 15-bp internal deletion mutants of the human PC distal promoter were generated and used in transient transfections in INS-1 832/13 insulinoma and HEK293T (kidney cell lines. The results indicated that positions -340 to -315 of the human PC distal promoter serve as (an activator element(s for cell-specific transcription factor, while the CCAAT box at -71/-67, a binding site for nuclear factor Y (NF-Y, as well as a GC box at -54/-39 of the human PC distal promoter act as activator sequences for basal transcription.

Are genetic variants in the platelet-derived growth factor [beta] gene associated with chronic pancreatitis?

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Muddana, Venkata; Park, James; Lamb, Janette; Yadav, Dhiraj; Papachristou, Georgios I; Hawes, Robert H; Brand, Randall; Slivka, Adam; Whitcomb, David C

2010-11-01

Platelet-derived growth factor [beta] (PDGF-[beta]) is a major signal in proliferation and matrix synthesis through activated pancreatic stellate cells, leading to fibrosis of the pancreas. Recurrent acute pancreatitis (RAP) seems to predispose to chronic pancreatitis (CP) in some patients but not others. We tested the hypothesis that 2 known PDGF-[beta] polymorphisms are associated with progression from RAP to CP. We also tested the hypothesis that PDGF-[beta] polymorphisms in combination with environmental risk factors such as alcohol and smoking are associated with CP. Three hundred eighty-two patients with CP (n = 176) and RAP (n = 206) and 251 controls were evaluated. Platelet-derived growth factor [beta] polymorphisms +286 A/G (rs#1800818) seen in 5'-UTR and +1135 A/C (rs#1800817) in first intron were genotyped using single-nucleotide polymorphism polymerase chain reaction approach and confirmed by DNA sequencing. The genotypic frequencies for PDGF-[beta] polymorphisms in positions +286 and +1135 were found to be similar in controls and patients with RAP and CP. There was no difference in genotypic frequencies among RAP, CP, and controls in subjects in the alcohol and smoking subgroups. Known variations in the PDGF-[beta] gene do not have a significant effect on promoting or preventing fibrogenesis in pancreatitis. Further evaluation of this important pathway is warranted.

PPAR gamma 2 prevents lipotoxicity by controlling adipose tissue expandability and peripheral lipid metabolism.

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Gema Medina-Gomez

2007-04-01

Full Text Available Peroxisome proliferator activated receptor gamma 2 (PPARg2 is the nutritionally regulated isoform of PPARg. Ablation of PPARg2 in the ob/ob background, PPARg2(-/- Lep(ob/Lep(ob (POKO mouse, resulted in decreased fat mass, severe insulin resistance, beta-cell failure, and dyslipidaemia. Our results indicate that the PPARg2 isoform plays an important role, mediating adipose tissue expansion in response to positive energy balance. Lipidomic analyses suggest that PPARg2 plays an important antilipotoxic role when induced ectopically in liver and muscle by facilitating deposition of fat as relatively harmless triacylglycerol species and thus preventing accumulation of reactive lipid species. Our data also indicate that PPARg2 may be required for the beta-cell hypertrophic adaptive response to insulin resistance. In summary, the PPARg2 isoform prevents lipotoxicity by (a promoting adipose tissue expansion, (b increasing the lipid-buffering capacity of peripheral organs, and (c facilitating the adaptive proliferative response of beta-cells to insulin resistance.

Enrichment of putative pancreatic progenitor cells from mice by sorting for prominin1 (CD133) and platelet-derived growth factor receptor beta.

Science.gov (United States)

Hori, Yuichi; Fukumoto, Miki; Kuroda, Yoshikazu

2008-11-01

Success in islet transplantation-based therapies for type 1 diabetes mellitus and an extreme shortage of pancreatic islets have motivated recent efforts to develop renewable sources of islet-replacement tissue. Although pancreatic progenitor cells hold a promising potential, only a few attempts have been made at the prospective isolation of pancreatic stem/progenitor cells, because of the lack of specific markers and the development of effective cell culture methods. We found that prominin1 (also known as CD133) recognized the undifferentiated epithelial cells, whereas platelet-derived growth factor receptor beta (PDGFRbeta) was expressed on the mesenchymal cells in the mouse embryonic pancreas. We then developed an isolation method for putative stem/progenitor cells by flow cytometric cell sorting and characterized their potential for differentiation to pancreatic tissue using both in vitro and in vivo protocols. Flow cytometry and the subsequent reverse transcription-polymerase chain reaction and microarray analysis revealed pancreatic epithelial progenitor cells to be highly enriched in the prominin1(high)PDGFRbeta(-) cell population. During in vivo differentiation, these cell populations were able to differentiate into endocrine, exocrine, and ductal tissues, including the formation of an insulin-producing cell cluster. We established the prospective isolation of putative pancreatic epithelial progenitor cells by sorting for prominin1 and PDGFRbeta. Since this strategy is based on the cell surface markers common to human and rodents, these findings may lead to the development of new strategies to derive transplantable islet-replacement tissues from human pancreatic stem/progenitor cells. Disclosure of potential conflicts of interest is found at the end of this article.

Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells.

Science.gov (United States)

D'Amour, Kevin A; Bang, Anne G; Eliazer, Susan; Kelly, Olivia G; Agulnick, Alan D; Smart, Nora G; Moorman, Mark A; Kroon, Evert; Carpenter, Melissa K; Baetge, Emmanuel E

2006-11-01

Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor--en route to cells that express endocrine hormones. The hES cell-derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal beta-cells, they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Production of these hES cell-derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy.

Lipidomic data on lipid droplet triglyceride remodelling associated with protection of breast cancer cells from lipotoxic stress

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Eva Jarc

2018-06-01

Full Text Available The data presented here is related to the research article entitled “Lipid droplets induced by secreted phospholipase A2 and unsaturated fatty acids protect breast cancer cells from nutrient and lipotoxic stress” by E. Jarc et al., Biochim. Biophys. Acta 1863 (2018 247–265. Elevated uptake of unsaturated fatty acids and lipid droplet accumulation are characteristic of aggressive cancer cells and have been associated with the cellular stress response. The present study provides lipidomic data on the triacylglycerol (TAG and phosphatidylcholine (PC composition of MDA-MB-231 breast cancer cells exposed to docosahexaenoic acid (DHA; 22:6, ω-3. Datasets provide information on the changes in lipid composition induced by depletion of adipose triglyceride lipase (ATGL and by exogenous addition of secreted phospholipase A2 (sPLA2 in DHA-treated cells. The presented alterations in lipid composition, mediated by targeting lipid droplet biogenesis and lipolysis, are associated with protection from lipotoxicity and allow further investigation into the role of lipid droplets in the resistance of cancer cells to lipotoxic stress. Keywords: Lipid droplets, Lipidomics, Adipose triglyceride lipase, Polyunsaturated fatty acid, Cancer, Phospholipase A2

Progenitor potential of nkx6.1-expressing cells throughout zebrafish life and during beta cell regeneration.

Science.gov (United States)

Ghaye, Aurélie P; Bergemann, David; Tarifeño-Saldivia, Estefania; Flasse, Lydie C; Von Berg, Virginie; Peers, Bernard; Voz, Marianne L; Manfroid, Isabelle

2015-09-02

In contrast to mammals, the zebrafish has the remarkable capacity to regenerate its pancreatic beta cells very efficiently. Understanding the mechanisms of regeneration in the zebrafish and the differences with mammals will be fundamental to discovering molecules able to stimulate the regeneration process in mammals. To identify the pancreatic cells able to give rise to new beta cells in the zebrafish, we generated new transgenic lines allowing the tracing of multipotent pancreatic progenitors and endocrine precursors. Using novel bacterial artificial chromosome transgenic nkx6.1 and ascl1b reporter lines, we established that nkx6.1-positive cells give rise to all the pancreatic cell types and ascl1b-positive cells give rise to all the endocrine cell types in the zebrafish embryo. These two genes are initially co-expressed in the pancreatic primordium and their domains segregate, not as a result of mutual repression, but through the opposite effects of Notch signaling, maintaining nkx6.1 expression while repressing ascl1b in progenitors. In the adult zebrafish, nkx6.1 expression persists exclusively in the ductal tree at the tip of which its expression coincides with Notch active signaling in centroacinar/terminal end duct cells. Tracing these cells reveals that they are able to differentiate into other ductal cells and into insulin-expressing cells in normal (non-diabetic) animals. This capacity of ductal cells to generate endocrine cells is supported by the detection of ascl1b in the nkx6.1:GFP ductal cell transcriptome. This transcriptome also reveals, besides actors of the Notch and Wnt pathways, several novel markers such as id2a. Finally, we show that beta cell ablation in the adult zebrafish triggers proliferation of ductal cells and their differentiation into insulin-expressing cells. We have shown that, in the zebrafish embryo, nkx6.1+ cells are bona fide multipotent pancreatic progenitors, while ascl1b+ cells represent committed endocrine precursors. In

Mechanisms of pancreatic beta-cell growth and regeneration

DEFF Research Database (Denmark)

Nielsen, Jens Høiriis

1989-01-01

Information about the mechanism of beta-cell growth and regeneration may be obtained by studies of insulinoma cells. In the present study the growth and function of the rat insulinoma cell lines RINm5F and 5AH were evaluated by addition of serum, hormones, and growth factors. It was found...... of insulin mRNA content showed that the insulinoma cells only contained about 2% of that of normal rat beta-cells. These results are discussed in relation to the role of growth factors, oncogenes, and differentiation in the growth and regeneration of beta-cells....... that transferrin is the only obligatory factor whereas growth hormone, epidermal growth factor, fibroblast growth factor, and TRH had modulating effects. A heat-labile heparin binding serum factor which stimulated thymidine incorporation but not cell proliferation was demonstrated in human serum. Measurements...

Lipotoxic effect of p21 on free fatty acid-induced steatosis in L02 cells.

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Jie-wei Wang

Full Text Available Nonalcoholic fatty liver disease (NAFLD is increasingly regarded as a hepatic manifestation of metabolic syndrome. Though with high prevalence, the mechanism is poorly understood. This study aimed to investigate the effects of p21 on free fatty acid (FFA-induced steatosis in L02 cells. We therefore analyzed the L02 cells with MG132 and siRNA treatment for different expression of p21 related to lipid accumulation and lipotoxicity. Cellular total lipid was stained by Oil Red O, while triglyceride content, cytotoxicity assays, lipid peroxidation markers and anti-oxidation levels were measured by enzymatic kits. Treatment with 1 mM FFA for 48 hr induced magnificent intracellular lipid accumulation and increased oxidative stress in p21 overload L02 cells compared to that in p21 knockdown L02 cells. By increasing oxidative stress and peroxidation, p21 accelerates FFA-induced lipotoxic effect in L02 cells and might provide information about potentially new targets for drug development and treatments of NAFLD.

SIRT6-mediated transcriptional suppression of Txnip is critical for pancreatic beta cell function and survival in mice.

Science.gov (United States)

Qin, Kunhua; Zhang, Ning; Zhang, Zhao; Nipper, Michael; Zhu, Zhenxin; Leighton, Jake; Xu, Kexin; Musi, Nicolas; Wang, Pei

2018-04-01

Better understanding of how genetic and epigenetic components control beta cell differentiation and function is key to the discovery of novel therapeutic approaches to prevent beta cell dysfunction and failure in the progression of type 2 diabetes. Our goal was to elucidate the role of histone deacetylase sirtuin 6 (SIRT6) in beta cell development and homeostasis. Sirt6 endocrine progenitor cell conditional knockout and beta cell-specific knockout mice were generated using the Cre-loxP system. Mice were assayed for islet morphology, glucose tolerance, glucose-stimulated insulin secretion and susceptibility to streptozotocin. Transcriptional regulatory functions of SIRT6 in primary islets were evaluated by RNA-Seq analysis. Reverse transcription-quantitative (RT-q)PCR and immunoblot were used to verify and investigate the gene expression changes. Chromatin occupancies of SIRT6, H3K9Ac, H3K56Ac and active RNA polymerase II were evaluated by chromatin immunoprecipitation. Deletion of Sirt6 in pancreatic endocrine progenitor cells did not affect endocrine morphology, beta cell mass or insulin production but did result in glucose intolerance and defective glucose-stimulated insulin secretion in mice. Conditional deletion of Sirt6 in adult beta cells reproduced the insulin secretion defect. Loss of Sirt6 resulted in aberrant upregulation of thioredoxin-interacting protein (TXNIP) in beta cells. SIRT6 deficiency led to increased acetylation of histone H3 lysine residue at 9 (H3K9Ac), acetylation of histone H3 lysine residue at 56 (H3K56Ac) and active RNA polymerase II at the promoter region of Txnip. SIRT6-deficient beta cells exhibited a time-dependent increase in H3K9Ac, H3K56Ac and TXNIP levels. Finally, beta cell-specific SIRT6-deficient mice showed increased sensitivity to streptozotocin. Our results reveal that SIRT6 suppresses Txnip expression in beta cells via deacetylation of histone H3 and plays a critical role in maintaining beta cell function and viability

Positron emission tomography study on pancreatic somatostatin receptors in normal and diabetic rats with {sup 68}Ga-DOTA-octreotide: A potential PET tracer for beta cell mass measurement

Energy Technology Data Exchange (ETDEWEB)

Sako, Takeo [Division of Bio-function Dynamics Imaging, RIKEN Center for Life Science Technologies, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047 (Japan); Division of Molecular Imaging, Institute of Biomedical Research and Innovation, 2-2 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047 (Japan); Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017 (Japan); Hasegawa, Koki; Nishimura, Mie; Kanayama, Yousuke; Wada, Yasuhiro; Hayashinaka, Emi; Cui, Yilong; Kataoka, Yosky [Division of Bio-function Dynamics Imaging, RIKEN Center for Life Science Technologies, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047 (Japan); Senda, Michio [Division of Bio-function Dynamics Imaging, RIKEN Center for Life Science Technologies, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047 (Japan); Division of Molecular Imaging, Institute of Biomedical Research and Innovation, 2-2 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047 (Japan); Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017 (Japan); Watanabe, Yasuyoshi, E-mail: yywata@riken.jp [Division of Bio-function Dynamics Imaging, RIKEN Center for Life Science Technologies, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047 (Japan)

2013-12-06

Highlights: •PET images showed high uptake of {sup 68}Ga-DOTA-octreotide in the normal pancreas. •{sup 68}Ga-DOTA-octreotide specifically binds to somatostatin receptors in the pancreas. •The pancreatic uptake of {sup 68}Ga-DOTA-octreotide was decreased in the diabetic rats. •{sup 68}Ga-DOTA-octreotide could be a candidate PET probe to measure the beta cell mass. -- Abstract: Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia, and the loss or dysfunction of pancreatic beta cells has been reported before the appearance of clinical symptoms and hyperglycemia. To evaluate beta cell mass (BCM) for improving the detection and treatment of DM at earlier stages, we focused on somatostatin receptors that are highly expressed in the pancreatic beta cells, and developed a positron emission tomography (PET) probe derived from octreotide, a metabolically stable somatostatin analog. Octreotide was conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a chelating agent, and labeled with {sup 68}Gallium ({sup 68}Ga). After intravenous injection of {sup 68}Ga-DOTA-octreotide, a 90-min emission scan of the abdomen was performed in normal and DM model rats. The PET studies showed that {sup 68}Ga-DOTA-octreotide radioactivity was highly accumulated in the pancreas of normal rats and that the pancreatic accumulation was significantly reduced in the rats administered with an excess amount of unlabeled octreotide or after treatment with streptozotocin, which was used for the chemical induction of DM in rats. These results were in good agreement with the ex vivo biodistribution data. These results indicated that the pancreatic accumulation of {sup 68}Ga-DOTA-octreotide represented specific binding to the somatostatin receptors and reflected BCM. Therefore, PET imaging with {sup 68}Ga-DOTA-octreotide could be a potential tool for evaluating BCM.

Pancreatic beta cells express two autoantigenic forms of glutamic acid decarboxylase, a 65-kDa hydrophilic form and a 64-kDa amphiphilic form which can be both membrane-bound and soluble

DEFF Research Database (Denmark)

Christgau, S; Schierbeck, H; Aanstoot, H J

1991-01-01

The 64-kDa pancreatic beta-cell autoantigen, which is a target of autoantibodies associated with early as well as progressive stages of beta-cell destruction, resulting in insulin-dependent diabetes (IDDM) in humans, has been identified as the gamma-aminobutyric acid-synthesizing enzyme glutamic...... acid decarboxylase. We have identified two autoantigenic forms of this protein in rat pancreatic beta-cells, a Mr 65,000 (GAD65) hydrophilic and soluble form of pI 6.9-7.1 and a Mr 64,000 (GAD64) component of pI 6.7. GAD64 is more abundant than GAD65 and has three distinct forms with regard to cellular...

The putative imidazoline receptor agonist, harmane, promotes intracellular calcium mobilisation in pancreatic beta-cells.

Science.gov (United States)

Squires, Paul E; Hills, Claire E; Rogers, Gareth J; Garland, Patrick; Farley, Sophia R; Morgan, Noel G

2004-10-06

beta-Carbolines (including harmane and pinoline) stimulate insulin secretion by a mechanism that may involve interaction with imidazoline I(3)-receptors but which also appears to be mediated by actions that are additional to imidazoline receptor agonism. Using the MIN6 beta-cell line, we now show that both the imidazoline I(3)-receptor agonist, efaroxan, and the beta-carboline, harmane, directly elevate cytosolic Ca(2+) and increase insulin secretion but that these responses display different characteristics. In the case of efaroxan, the increase in cytosolic Ca(2+) was readily reversible, whereas, with harmane, the effect persisted beyond removal of the agonist and resulted in the development of a repetitive train of Ca(2+)-oscillations whose frequency, but not amplitude, was concentration-dependent. Initiation of the Ca(2+)-oscillations by harmane was independent of extracellular calcium but was sensitive to both dantrolene and high levels (20 mM) of caffeine, suggesting the involvement of ryanodine receptor-gated Ca(2+)-release. The expression of ryanodine receptor-1 and ryanodine receptor-2 mRNA in MIN6 cells was confirmed using reverse transcription-polymerase chain reaction (RT-PCR) and, since low concentrations of caffeine (1 mM) or thimerosal (10 microM) stimulated increases in [Ca(2+)](i), we conclude that ryanodine receptors are functional in these cells. Furthermore, the increase in insulin secretion induced by harmane was attenuated by dantrolene, consistent with the involvement of ryanodine receptors in mediating this response. By contrast, the smaller insulin secretory response to efaroxan was unaffected by dantrolene. Harmane-evoked changes in cytosolic Ca(2+) were maintained by nifedipine-sensitive Ca(2+)-influx, suggesting the involvement of L-type voltage-gated Ca(2+)-channels. Taken together, these data imply that harmane may interact with ryanodine receptors to generate sustained Ca(2+)-oscillations in pancreatic beta-cells and that this effect

Leucine metabolism in regulation of insulin secretion from pancreatic beta cells

OpenAIRE

Yang, Jichun; Chi, Yujing; Burkhardt, Brant R.; Guan, Youfei; Wolf, Bryan A

2010-01-01

Leucine, a the branched-chain amino acids that must be supplied in daily diet, plays an important role in controlling protein synthesis and regulating cell metabolism in various cell types. In pancreatic β cells, leucine acutely stimulates insulin secretion by serving as both metabolic fuel and allosteric activator of glutamate dehydrogenase to enhance glutaminolysis. Leucine has also been shown to regulate gene transcription and protein synthesis in pancreatic islet β cells via both mTOR-dep...

Progress in molecular nuclear medicine imaging of pancreatic beta cells

International Nuclear Information System (INIS)

Wu Haifei; Yin Hongyan; Liu Shuai; Zhang Yifan

2010-01-01

Diabetes mellitus is a common and frequently occurring disease which seriously threaten the health of human beings. Type 1 and type 2 diabetes respectively results from being destroyed and insufficient beta-cell mass. The associated symptoms appear until 50%-60% decrease of beta-cell mass. Because pancreas is deeply located in the body, with few beta-cell mass, the current methods of clinical diagnosis are invasive and late. So diagnosis of metabolism disease of beta-cell early non-invasively becomes more and more popular, imaging diagnosis of diabetes mellitus becomes the focus of researches, but how to estimate the mass of beta-cell still an important subject in imaging technology. (authors)

The islet beta-cell: fuel responsive and vulnerable.

Science.gov (United States)

Nolan, Christopher J; Prentki, Marc

2008-10-01

The pancreatic beta-cell senses blood nutrient levels and is modulated by neurohormonal signals so that it secretes insulin according to the need of the organism. Nutrient sensing involves marked metabolic activation, resulting in the production of coupling signals that promote insulin biosynthesis and secretion. The beta-cell's high capacity for nutrient sensing, however, necessitates reduced protection to nutrient toxicity. This potentially explains why in susceptible individuals, chronic fuel surfeit results in beta-cell failure and type 2 diabetes. Here we discuss recent insights into first, the biochemical basis of beta-cell signaling in response to glucose, amino acids and fatty acids, and second, beta-cell nutrient detoxification. We emphasize the emerging role of glycerolipid/fatty acid cycling in these processes.

Fetal pancreatic beta-cell function in pregnancies complicated by maternal diabetes mellitus: relationship to fetal acidemia and macrosomia.

Science.gov (United States)

Salvesen, D R; Brudenell, J M; Proudler, A J; Crook, D; Nicolaides, K H

1993-05-01

Our purpose was to investigate the relationship between fetal pancreatic beta-cell function and fetal acidemia and macrosomia in pregnancies complicated by maternal diabetes mellitus. A cross-sectional study at the Harris Birthright Research Centre for Fetal Medicine, London, was performed. In 32 pregnancies complicated by maternal diabetes mellitus cordocentesis was performed at 36 to 39 weeks' gestation for the measurement of umbilical venous blood pH, PO2, PCO2, lactate, and glucose concentration; plasma insulin immunoreactivity; and insulin/glucose ratio. A reference range for plasma insulin and insulin/glucose ratio was constructed by studying fetal blood samples from 80 women who did not have diabetes mellitus. Mean umbilical venous blood pH was significantly lower and plasma insulin immunoreactivity and insulin/glucose ratio were significantly higher than the appropriate normal mean for gestation. There were significant associations between (1) maternal and fetal blood glucose concentrations (r = 0.95, p < 0.0001), (2) fetal blood glucose and plasma insulin immunoreactivity (r = 0.57, p < 0.01), (3) fetal plasma insulin immunoreactivity and blood pH (r = -0.39, p < 0.05), and (4) fetal insulin/glucose ratio and degree of macrosomia (r = 0.76, p < 0.0001). Fetal pancreatic beta-cell hyperplasia is implicated in the pathogenesis of both fetal acidemia and macrosomia.

Positron emission tomography study on pancreatic somatostatin receptors in normal and diabetic rats with 68Ga-DOTA-octreotide: a potential PET tracer for beta cell mass measurement.

Science.gov (United States)

Sako, Takeo; Hasegawa, Koki; Nishimura, Mie; Kanayama, Yousuke; Wada, Yasuhiro; Hayashinaka, Emi; Cui, Yilong; Kataoka, Yosky; Senda, Michio; Watanabe, Yasuyoshi

2013-12-06

Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia, and the loss or dysfunction of pancreatic beta cells has been reported before the appearance of clinical symptoms and hyperglycemia. To evaluate beta cell mass (BCM) for improving the detection and treatment of DM at earlier stages, we focused on somatostatin receptors that are highly expressed in the pancreatic beta cells, and developed a positron emission tomography (PET) probe derived from octreotide, a metabolically stable somatostatin analog. Octreotide was conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a chelating agent, and labeled with (68)Gallium ((68)Ga). After intravenous injection of (68)Ga-DOTA-octreotide, a 90-min emission scan of the abdomen was performed in normal and DM model rats. The PET studies showed that (68)Ga-DOTA-octreotide radioactivity was highly accumulated in the pancreas of normal rats and that the pancreatic accumulation was significantly reduced in the rats administered with an excess amount of unlabeled octreotide or after treatment with streptozotocin, which was used for the chemical induction of DM in rats. These results were in good agreement with the ex vivo biodistribution data. These results indicated that the pancreatic accumulation of (68)Ga-DOTA-octreotide represented specific binding to the somatostatin receptors and reflected BCM. Therefore, PET imaging with (68)Ga-DOTA-octreotide could be a potential tool for evaluating BCM. Copyright © 2013 Elsevier Inc. All rights reserved.

Role of transcription factor KLF11 and its diabetes-associated gene variants in pancreatic beta cell function

DEFF Research Database (Denmark)

Neve, Bernadette; Fernandez-Zapico, Martin E; Ashkenazi-Katalan, Vered

2005-01-01

in beta cells. Genetic analysis of the KLF11 gene revealed two rare variants (Ala347Ser and Thr220Met) that segregate with diabetes in families with early-onset type 2 diabetes, and significantly impair its transcriptional activity. In addition, analysis of 1,696 type 2 diabetes mellitus and 1......,776 normoglycemic subjects show a frequent polymorphic Gln62Arg variant that significantly associates with type 2 diabetes mellitus in North European populations (OR = 1.29, P = 0.00033). Moreover, this variant alters the corepressor mSin3A-binding activity of KLF11, impairs the activation of the insulin promoter...... and shows lower levels of insulin expression in pancreatic beta cells. In addition, subjects carrying the Gln62Arg allele show decreased plasma insulin after an oral glucose challenge. Interestingly, all three nonsynonymous KLF11 variants show increased repression of the catalase 1 promoter, suggesting...

Pancreatic differentiation of Pdx1-GFP reporter mouse induced pluripotent stem cells.

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Porciuncula, Angelo; Kumar, Anujith; Rodriguez, Saray; Atari, Maher; Araña, Miriam; Martin, Franz; Soria, Bernat; Prosper, Felipe; Verfaillie, Catherine; Barajas, Miguel

2016-12-01

Efficient induction of defined lineages in pluripotent stem cells constitutes the determinant step for the generation of therapeutically relevant replacement cells to potentially treat a wide range of diseases, including diabetes. Pancreatic differentiation has remained an important challenge in large part because of the need to differentiate uncommitted pluripotent stem cells into highly specialized hormone-secreting cells, which has been shown to require a developmentally informed step-by-step induction procedure. Here, in the framework of using induced pluripotent stem cells (iPSCs) to generate pancreatic cells for pancreatic diseases, we have generated and characterized iPSCs from Pdx1-GFP transgenic mice. The use of a GFP reporter knocked into the endogenous Pdx1 promoter allowed us to monitor pancreatic induction based on the expression of Pdx1, a pancreatic master transcription factor, and to isolate a pure Pdx1-GFP + population for downstream applications. Differentiated cultures timely expressed markers specific to each stage and end-stage progenies acquired a rather immature beta-cell phenotype, characterized by polyhormonal expression even among cells highly expressing the Pdx1-GFP reporter. Our findings highlight the utility of employing a fluorescent protein reporter under the control of a master developmental gene in order to devise novel differentiation protocols for relevant cell types for degenerative diseases such as pancreatic beta cells for diabetes. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

On the coherent behavior of pancreatic beta cell clusters

Energy Technology Data Exchange (ETDEWEB)

Loppini, Alessandro, E-mail: a.loppini@unicampus.it [Nonlinear Physics and Mathematical Modeling Lab, University Campus Bio-Medico, Via A. del Portillo 21, I-00128 Rome (Italy); Capolupo, Antonio, E-mail: capolupo@sa.infn.it [Physics Department, University of Salerno, Fisciano, 84084 (Italy); Cherubini, Christian, E-mail: c.cherubini@unicampus.it [Nonlinear Physics and Mathematical Modeling Lab, University Campus Bio-Medico, Via A. del Portillo 21, I-00128 Rome (Italy); International Center for Relativistic Astrophysics, University Campus Bio-Medico, Via A. del Portillo 21, I-00128, Rome (Italy); Gizzi, Alessio, E-mail: a.gizzi@unicampus.it [Nonlinear Physics and Mathematical Modeling Lab, University Campus Bio-Medico, Via A. del Portillo 21, I-00128 Rome (Italy); Bertolaso, Marta, E-mail: m.bertolaso@unicampus.it [Faculty of Engineering and Institute of Philosophy of Scientific and Technological Practice, University Campus Bio-Medico, Via A. del Portillo 21, I-00128 Rome (Italy); Filippi, Simonetta, E-mail: s.filippi@unicampus.it [Nonlinear Physics and Mathematical Modeling Lab, University Campus Bio-Medico, Via A. del Portillo 21, I-00128 Rome (Italy); International Center for Relativistic Astrophysics, University Campus Bio-Medico, Via A. del Portillo 21, I-00128, Rome (Italy); Vitiello, Giuseppe, E-mail: vitiello@sa.infn.it [Physics Department, University of Salerno, Fisciano, 84084 (Italy)

2014-09-12

Beta cells in pancreas represent an example of coupled biological oscillators which via communication pathways, are able to synchronize their electrical activity, giving rise to pulsatile insulin release. In this work we numerically analyze scale free self-similarity features of membrane voltage signal power density spectrum, through a stochastic dynamical model for beta cells in the islets of Langerhans fine tuned on mouse experimental data. Adopting the algebraic approach of coherent state formalism, we show how coherent molecular domains can arise from proper functional conditions leading to a parallelism with “phase transition” phenomena of field theory. - Highlights: • Beta cells in pancreas are coupled oscillators able to synchronize their activity. • We analyze scale free self-similarity features for beta cells. • We adopt the algebraic approach of coherent state formalism. • We show that coherent molecular domains arise from functional conditions.

The Importance of REST for Development and Function of Beta Cells

DEFF Research Database (Denmark)

Martin, David; Grapin-Botton, Anne

2017-01-01

that are crucial for both neuronal and pancreatic endocrine function, through the recruitment of multiple transcriptional and epigenetic co-regulators. REST targets include genes encoding transcription factors, proteins involved in exocytosis, synaptic transmission or ion channeling, and non-coding RNAs. REST......Beta cells are defined by the genes they express, many of which are specific to this cell type, and ensure a specific set of functions. Beta cells are also defined by a set of genes they should not express (in order to function properly), and these genes have been called forbidden genes. Among...... these, the transcriptional repressor RE-1 Silencing Transcription factor (REST) is expressed in most cells of the body, excluding most populations of neurons, as well as pancreatic beta and alpha cells. In the cell types where it is expressed, REST represses the expression of hundreds of genes...

Role of bone marrow cells in the development of pancreatic fibrosis in a rat model of pancreatitis induced by a choline-deficient/ethionine-supplemented diet

Energy Technology Data Exchange (ETDEWEB)

Akita, Shingo; Kubota, Koji [Department of Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Kobayashi, Akira, E-mail: kbys@shinshu-u.ac.jp [Department of Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Misawa, Ryosuke; Shimizu, Akira; Nakata, Takenari; Yokoyama, Takahide [Department of Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Takahashi, Masafumi [Center for Molecular Medicine Division of Bioimaging Sciences, Jichi Medical University, 3311-1 Yakushiji, Shimono, Tochigi 329-0498 (Japan); Miyagawa, Shinichi [Department of Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan)

2012-04-20

Highlights: Black-Right-Pointing-Pointer BMC-derived PSCs play a role in a rat CDE diet-induced pancreatitis model. Black-Right-Pointing-Pointer BMC-derived PSCs contribute mainly to the early stage of pancreatic fibrosis. Black-Right-Pointing-Pointer BMC-derived activated PSCs can produce PDGF and TGF {beta}1. -- Abstract: Bone marrow cell (BMC)-derived myofibroblast-like cells have been reported in various organs, including the pancreas. However, the contribution of these cells to pancreatic fibrosis has not been fully discussed. The present study examined the possible involvement of pancreatic stellate cells (PSCs) originating from BMCs in the development of pancreatic fibrosis in a clinically relevant rat model of acute pancreatitis induced by a choline-deficient/ethionine-supplemented (CDE) diet. BMCs from female transgenic mice ubiquitously expressing green fluorescent protein (GFP) were transplanted into lethally irradiated male rats. Once chimerism was established, acute pancreatitis was induced by a CDE diet. Chronological changes in the number of PSCs originating from the donor BMCs were examined using double immunofluorescence for GFP and markers for PSCs, such as desmin and alpha smooth muscle actin ({alpha}SMA), 1, 3 and 8 weeks after the initiation of CDE feeding. We also used immunohistochemical staining to evaluate whether the PSCs from the BMCs produce growth factors, such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF) {beta}1. The percentage of BMC-derived activated PSCs increased significantly, peaking after 1 week of CDE treatment (accounting for 23.3 {+-} 0.9% of the total population of activated PSCs) and then decreasing. These cells produced both PDGF and TGF{beta}1 during the early stage of pancreatic fibrosis. Our results suggest that PSCs originating from BMCs contribute mainly to the early stage of pancreatic injury, at least in part, by producing growth factors in a rat CDE diet-induced pancreatitis model.

Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene

DEFF Research Database (Denmark)

Efrat, S; Linde, S; Kofod, Hans

1988-01-01

Three pancreatic beta-cell lines have been established from insulinomas derived from transgenic mice carrying a hybrid insulin-promoted simian virus 40 tumor antigen gene. The beta tumor cell (beta TC) lines maintain the features of differentiated beta cells for about 50 passages in culture. The ...... both to immortalize a rare cell type and to provide a selection for the maintenance of its differentiated phenotype....

Chronology of endocrine differentiation and beta-cell neogenesis.

Science.gov (United States)

Miyatsuka, Takeshi

2016-01-01

Diabetes is a chronic and incurable disease, which results from absolute or relative insulin insufficiency. Therefore, pancreatic beta cells, which are the only type of cell that expresses insulin, is considered to be a potential target for the cure of diabetes. Although the findings regarding beta-cell neogenesis during pancreas development have been exploited to induce insulin-producing cells from non-beta cells, there are still many hurdles towards generating fully functional beta cells that can produce high levels of insulin and respond to physiological signals. To overcome these problems, a solid understanding of pancreas development and beta-cell formation is required, and several mouse models have been developed to reveal the unique features of each endocrine cell type at distinct developmental time points. Here I review our understanding of pancreas development and endocrine differentiation focusing on recent progresses in improving temporal cell labeling in vivo.

Insulin-producing cells generated from dedifferentiated human pancreatic beta cells expanded in vitro.

Directory of Open Access Journals (Sweden)

Holger A Russ

Full Text Available Expansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT. Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells.Redifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2 using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation.These findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug

Fetal and neonatal nicotine exposure in Wistar rats causes progressive pancreatic mitochondrial damage and beta cell dysfunction.

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Jennifer E Bruin

Full Text Available Nicotine replacement therapy (NRT is currently recommended as a safe smoking cessation aid for pregnant women. However, fetal and neonatal nicotine exposure in rats causes mitochondrial-mediated beta cell apoptosis at weaning, and adult-onset dysglycemia, which we hypothesize is related to progressive mitochondrial dysfunction in the pancreas. Therefore in this study we examined the effect of fetal and neonatal exposure to nicotine on pancreatic mitochondrial structure and function during postnatal development. Female Wistar rats were given saline (vehicle control or nicotine bitartrate (1 mg/kg/d via subcutaneous injection for 2 weeks prior to mating until weaning. At 3-4, 15 and 26 weeks of age, oral glucose tolerance tests were performed, and pancreas tissue was collected for electron microscopy, enzyme activity assays and islet isolation. Following nicotine exposure mitochondrial structural abnormalities were observed beginning at 3 weeks and worsened with advancing age. Importantly the appearance of these structural defects in nicotine-exposed animals preceded the onset of glucose intolerance. Nicotine exposure also resulted in significantly reduced pancreatic respiratory chain enzyme activity, degranulation of beta cells, elevated islet oxidative stress and impaired glucose-stimulated insulin secretion compared to saline controls at 26 weeks of age. Taken together, these data suggest that maternal nicotine use during pregnancy results in postnatal mitochondrial dysfunction that may explain, in part, the dysglycemia observed in the offspring from this animal model. These results clearly indicate that further investigation into the safety of NRT use during pregnancy is warranted.

Microvesicles released from fat-laden cells promote activation of hepatocellular NLRP3 inflammasome: A pro-inflammatory link between lipotoxicity and non-alcoholic steatohepatitis.

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Stefania Cannito

Full Text Available Non-Alcoholic Fatty Liver Disease (NAFLD is a major form of chronic liver disease in the general population in relation to its high prevalence among overweight/obese individuals and patients with diabetes type II or metabolic syndrome. NAFLD can progress to steatohepatitis (NASH, fibrosis and cirrhosis and end-stage of liver disease but mechanisms involved are still incompletely characterized. Within the mechanisms proposed to mediate the progression of NAFLD, lipotoxicity is believed to play a major role. In the present study we provide data suggesting that microvesicles (MVs released by fat-laden cells undergoing lipotoxicity can activate NLRP3 inflammasome following internalization by either cells of hepatocellular origin or macrophages. Inflammasome activation involves NF-kB-mediated up-regulation of NLRP3, pro-caspase-1 and pro-Interleukin-1, then inflammasome complex formation and Caspase-1 activation leading finally to an increased release of IL-1β. Since the release of MVs from lipotoxic cells and the activation of NLRP3 inflammasome have been reported to occur in vivo in either clinical or experimental NASH, these data suggest a novel rational link between lipotoxicity and increased inflammatory response.

Regulation of Pancreatic β Cell Mass by Cross-Interaction between CCAAT Enhancer Binding Protein β Induced by Endoplasmic Reticulum Stress and AMP-Activated Protein Kinase Activity.

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Tomokazu Matsuda

Full Text Available During the development of type 2 diabetes, endoplasmic reticulum (ER stress leads to not only insulin resistance but also to pancreatic beta cell failure. Conversely, cell function under various stressed conditions can be restored by reducing ER stress by activating AMP-activated protein kinase (AMPK. However, the details of this mechanism are still obscure. Therefore, the current study aims to elucidate the role of AMPK activity during ER stress-associated pancreatic beta cell failure. MIN6 cells were loaded with 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR and metformin to assess the relationship between AMPK activity and CCAAT enhancer binding protein β (C/EBPβ expression levels. The effect of C/EBPβ phosphorylation on expression levels was also investigated. Vildagliptin and metformin were administered to pancreatic beta cell-specific C/EBPβ transgenic mice to investigate the relationship between C/EBPβ expression levels and AMPK activity in the pancreatic islets. When pancreatic beta cells are exposed to ER stress, the accumulation of the transcription factor C/EBPβ lowers the AMP/ATP ratio, thereby decreasing AMPK activity. In an opposite manner, incubation of MIN6 cells with AICAR or metformin activated AMPK, which suppressed C/EBPβ expression. In addition, administration of the dipeptidyl peptidase-4 inhibitor vildagliptin and metformin to pancreatic beta cell-specific C/EBPβ transgenic mice decreased C/EBPβ expression levels and enhanced pancreatic beta cell mass in proportion to the recovery of AMPK activity. Enhanced C/EBPβ expression and decreased AMPK activity act synergistically to induce ER stress-associated pancreatic beta cell failure.

[Relationship between the expression of beta-cat, cyclin D1 and c-myc and the occurance and biological behavior of pancreatic cancer].

Science.gov (United States)

Li, Yu-jun; Ji, Xiang-rui

2003-06-01

To study the relationship between the abnormal expression of beta-catenin (beta-cat) and the high expressions of cyclin D1 and c-myc and the occurance, proliferation, infiltration, metastasis and prognosis of pancreatic cancer, and to provide rational basis for the clinical diagnosis and treatment. Immunohistochemical PicTure trade mark was used to examine the expressions of beta-cat, cyclin D1 and c-myc in 47 cases of the cancerous tissue of pancreas, 12 cases of the pancreatic intraepithelial neoplasia and 10 cases of normal tissue of pancreas, respectively. Pancreatic cancer proliferation cell nuclear antigen (PCNA) was also tested as the index of the extent of proliferation of the pancreatic cancer. beta-cat was expressed normally in the 10 cases of the normal pancreatic tissue, while cyclin D1 and c-myc were negative. The expression rates of beta-cat, cyclin D1 and c-myc in the tissues of the pancreatic intraepithelial neoplasia and the pancreatic cancer had no significant difference [6/12 and 68.1% (32/47), 6/12 and 74.5% (35/47), 5/12 and 70.2% (33/47) respectively;P values were all more than 0.05]. The abnormal expression rate of beta-cat was significantly correlated to the metastasis of the pancreatic cancer and the one-year survival rate (both P 0.05). The expression rate of cyclin D1 was correlated with the proliferation of the pancreatic cancer and the extent of differentiation (both P 0.05). The expression rate of c-myc was not correlated with the size, the extent of proliferation, infiltration, metastasis, or one-year survival rate (both P > 0.05), but closely with the proliferation activity of the cancerous tissue of pancreas (P < 0.05). The abnormal expression of beta-cat and the high expressions of cyclin D1 and c-myc had a parallel relationship with the pancreatic intraepithelial neoplasia and pancreatic cancer (both P < 0.05, gamma = 1.000, 0.845, 0.437, 0.452). The abnormal expression of beta-cat activates cyclin D1 and c-myc, and results in the

Transgenic overexpression of active calcineurin in beta-cells results in decreased beta-cell mass and hyperglycemia.

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Ernesto Bernal-Mizrachi

2010-08-01

Full Text Available Glucose modulates beta-cell mass and function through an initial depolarization and Ca(2+ influx, which then triggers a number of growth regulating signaling pathways. One of the most important downstream effectors in Ca(2+ signaling is the calcium/Calmodulin activated serine threonine phosphatase, calcineurin. Recent evidence suggests that calcineurin/NFAT is essential for beta-cell proliferation, and that in its absence loss of beta-cells results in diabetes. We hypothesized that in contrast, activation of calcineurin might result in expansion of beta-cell mass and resistance to diabetes.To determine the role of activation of calcineurin signaling in the regulation of pancreatic beta-cell mass and proliferation, we created mice that expressed a constitutively active form of calcineurin under the insulin gene promoter (caCn(RIP. To our surprise, these mice exhibited glucose intolerance. In vitro studies demonstrated that while the second phase of Insulin secretion is enhanced, the overall insulin secretory response was conserved. Islet morphometric studies demonstrated decreased beta-cell mass suggesting that this was a major component responsible for altered Insulin secretion and glucose intolerance in caCn(RIP mice. The reduced beta-cell mass was accompanied by decreased proliferation and enhanced apoptosis.Our studies identify calcineurin as an important factor in controlling glucose homeostasis and indicate that chronic depolarization leading to increased calcineurin activity may contribute, along with other genetic and environmental factors, to beta-cell dysfunction and diabetes.

Planar Cell Polarity Controls Pancreatic Beta Cell Differentiation and Glucose Homeostasis

DEFF Research Database (Denmark)

Cortijo, Cedric; Gouzi, Mathieu; Tissir, Fadel

2012-01-01

glucose clearance. Loss of Celsr2 and 3 leads to a reduction of Jun phosphorylation in progenitors, which, in turn, reduces beta cell differentiation from endocrine progenitors. These results highlight the importance of the PCP pathway in cell differentiation in vertebrates. In addition, they reveal.......5 synchronously to apicobasal polarization of pancreas progenitors. Loss of function of the two PCP core components Celsr2 and Celsr3 shows that they control the differentiation of endocrine cells from polarized progenitors, with a prevalent effect on insulin-producing beta cells. This results in a decreased...

Pancreatic stellate cells enhance stem cell-like phenotypes in pancreatic cancer cells

International Nuclear Information System (INIS)

Hamada, Shin; Masamune, Atsushi; Takikawa, Tetsuya; Suzuki, Noriaki; Kikuta, Kazuhiro; Hirota, Morihisa; Hamada, Hirofumi; Kobune, Masayoshi; Satoh, Kennichi; Shimosegawa, Tooru

2012-01-01

Highlights: ► Pancreatic stellate cells (PSCs) promote the progression of pancreatic cancer. ► Pancreatic cancer cells co-cultured with PSCs showed enhanced spheroid formation. ► Expression of stem cell-related genes ABCG2, Nestin and LIN28 was increased. ► Co-injection of PSCs enhanced tumorigenicity of pancreatic cancer cells in vivo. ► This study suggested a novel role of PSCs as a part of the cancer stem cell niche. -- Abstract: The interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, is receiving increasing attention. There is accumulating evidence that PSCs promote the progression of pancreatic cancer by increasing cancer cell proliferation and invasion as well as by protecting them from radiation- and gemcitabine-induced apoptosis. Recent studies have identified that a portion of cancer cells, called “cancer stem cells”, within the entire cancer tissue harbor highly tumorigenic and chemo-resistant phenotypes, which lead to the recurrence after surgery or re-growth of the tumor. The mechanisms that maintain the “stemness” of these cells remain largely unknown. We hypothesized that PSCs might enhance the cancer stem cell-like phenotypes in pancreatic cancer cells. Indirect co-culture of pancreatic cancer cells with PSCs enhanced the spheroid-forming ability of cancer cells and induced the expression of cancer stem cell-related genes ABCG2, Nestin and LIN28. In addition, co-injection of PSCs enhanced tumorigenicity of pancreatic cancer cells in vivo. These results suggested a novel role of PSCs as a part of the cancer stem cell niche.

Lysine deacetylase inhibition prevents diabetes by chromatin-independent immunoregulation and beta-cell protection

NARCIS (Netherlands)

Christensen, D.P.; Gysemans, C.; Lundh, M.; Dahllof, M.S.; Noesgaard, D.; Schmidt, S.F.; Mandrup, S; Birkbak, N.; Workman, C.T.; Piemonti, L.; Blaabjerg, L.; Monzani, V.; Fossati, G.; Mascagni, P.; Paraskevas, S.; Aikin, R.A.; Billestrup, N.; Grunnet, L.G.; Dinarello, C.A.; Mathieu, C.; Mandrup-Poulsen, T.

2014-01-01

Type 1 diabetes is due to destruction of pancreatic beta-cells. Lysine deacetylase inhibitors (KDACi) protect beta-cells from inflammatory destruction in vitro and are promising immunomodulators. Here we demonstrate that the clinically well-tolerated KDACi vorinostat and givinostat revert diabetes

Development of biomarker specific of pancreatic beta cells (incretin radiolabelled) for image of beta functional mass in diabetic and obese: study in animal model; Desenvolvimento de biomarcador específico de células beta pancreáticas (incretina radiomarcada) para imagem da massa beta funcional em diabéticos e obesos: estudo em modelo animal

Energy Technology Data Exchange (ETDEWEB)

Seo, Daniele

2017-07-01

Increased prevalence of obesity worldwide, has become a vast concern, stimulating investigations focusing prevention and therapy of this condition. The association of type 2 diabetes or insulin resistance aggravates the prognosis of obesity. Even patients successfully submitted to bariatric or metabolic surgery, may not be cured of diabetes, as improvement of circulating values of glucose and insulin not necessarily reflects recovery of pancreatic beta cell mass. There is no consensus about how to estimate beta cell mass in vivo. Available tools suffer from low sensitivity and specificity, often being as well cumbersome and expensive. Radiolabeled incretins, such as glucagon-like-peptide 1 (GLP-1) analogs, seem to be promising options for the measurement of beta cell mass in diabetes and insulinoma. The objective of this study was the development of two conjugates of GLP-1 analog, radiolabeled with {sup 99m} Technetium, as a noninvasive imaging method for the estimation of pancreatic beta cell mass, in the presence of obesity. Animal models were selected, including hyperlipidic diet-induced obesity, diet restricted obesity, and as controls, alloxan diabetes. Results indicated that both radiotracers achieved over 97% radiochemical yield. The most successful product was {sup 99m}Tc-HYNIC-βAla-Exendin-4. Low beta cell mass uptake occurred in diet-induced obesity. Diet-restricted obesity, with substantial shedding of excess body weight, was followed by remarkable decrease of fasting blood glucose, however beta cell mass uptake was only mildly improved. Future studies are recommended in obesity, type 2 diabetes, and dieting, including bariatric and metabolic operations. (author)

Redox Homeostasis in Pancreatic beta Cells

Czech Academy of Sciences Publication Activity Database

Ježek, Petr; Dlasková, Andrea; Plecitá-Hlavatá, Lydie

2012-01-01

Roč. 2012, č. 2012 (2012), s. 932838 ISSN 1942-0900 R&D Projects: GA ČR(CZ) GAP302/10/0346; GA ČR(CZ) GPP304/10/P204 Institutional support: RVO:67985823 Keywords : beta cells * reactive oxygen species homeostasis * mitochondria Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 3.393, year: 2012

Carriers of loss-of-function mutations in EXT display impaired pancreatic beta-cell reserve due to smaller pancreas volume.

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Sophie J Bernelot Moens

Full Text Available Exotosin (EXT proteins are involved in the chain elongation step of heparan sulfate (HS biosynthesis, which is intricately involved in organ development. Loss of function mutations (LOF in EXT1 and EXT2 result in hereditary exostoses (HME. Interestingly, HS plays a role in pancreas development and beta-cell function, and genetic variations in EXT2 are associated with an increased risk for type 2 diabetes mellitus. We hypothesized that loss of function of EXT1 or EXT2 in subjects with hereditary multiple exostoses (HME affects pancreatic insulin secretion capacity and development. We performed an oral glucose tolerance test (OGTT followed by hyperglycemic clamps to investigate first-phase glucose-stimulated insulin secretion (GSIS in HME patients and age and gender matched non-affected relatives. Pancreas volume was assessed with magnetic resonance imaging (MRI. OGTT did not reveal significant differences in glucose disposal, but there was a markedly lower GSIS in HME subjects during hyperglycemic clamp (iAUC HME: 0.72 [0.46-1.16] vs. controls 1.53 [0.69-3.36] nmol·l-1·min-1, p<0.05. Maximal insulin response following arginine challenge was also significantly attenuated (iAUC HME: 7.14 [4.22-10.5] vs. controls 10.2 [7.91-12.70] nmol·l-1·min-1 p<0.05, indicative of an impaired beta-cell reserve. MRI revealed a significantly smaller pancreatic volume in HME subjects (HME: 72.0±15.8 vs. controls 96.5±26.0 cm3 p = 0.04. In conclusion, loss of function of EXT proteins may affect beta-cell mass and insulin secretion capacity in humans, and render subjects at a higher risk of developing type 2 diabetes when exposed to environmental risk factors.

Transcriptomic profiling of pancreatic alpha, beta and delta cell populations identifies delta cells as a principal target for ghrelin in mouse islets

DEFF Research Database (Denmark)

Adriaenssens, Alice E; Svendsen, Berit; Lam, Brian Y H

2016-01-01

cytometry and analysed by RNA sequencing. The role of the ghrelin receptor was validated by imaging delta cell calcium concentrations using islets with delta cell restricted expression of the calcium reporter GCaMP3, and in perfused mouse pancreases. RESULTS: A database was constructed of all genes...... expressed in alpha, beta and delta cells. The gene encoding the ghrelin receptor, Ghsr, was highlighted as being highly expressed and enriched in delta cells. Activation of the ghrelin receptor raised cytosolic calcium levels in primary pancreatic delta cells and enhanced somatostatin secretion in perfused...... pancreases, correlating with a decrease in insulin and glucagon release. The inhibition of insulin secretion by ghrelin was prevented by somatostatin receptor antagonism. CONCLUSIONS/INTERPRETATION: Our transcriptomic database of genes expressed in the principal islet cell populations will facilitate...

Pancreatic stellate cells promote epithelial-mesenchymal transition in pancreatic cancer cells

International Nuclear Information System (INIS)

Kikuta, Kazuhiro; Masamune, Atsushi; Watanabe, Takashi; Ariga, Hiroyuki; Itoh, Hiromichi; Hamada, Shin; Satoh, Kennichi; Egawa, Shinichi; Unno, Michiaki; Shimosegawa, Tooru

2010-01-01

Research highlights: → Recent studies have shown that pancreatic stellate cells (PSCs) promote the progression of pancreatic cancer. → Pancreatic cancer cells co-cultured with PSCs showed loose cell contacts and scattered, fibroblast-like appearance. → PSCs decreased the expression of epithelial markers but increased that of mesenchymal markers, along with increased migration. → This study suggests epithelial-mesenchymal transition as a novel mechanism by which PSCs contribute to the aggressive behavior of pancreatic cancer cells. -- Abstract: The interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, is receiving increasing attention. There is accumulating evidence that PSCs promote the progression of pancreatic cancer by increasing cancer cell proliferation and invasion as well as by protecting them from radiation- and gemcitabine-induced apoptosis. Because epithelial-mesenchymal transition (EMT) plays a critical role in the progression of pancreatic cancer, we hypothesized that PSCs promote EMT in pancreatic cancer cells. Panc-1 and SUIT-2 pancreatic cancer cells were indirectly co-cultured with human PSCs isolated from patients undergoing operation for pancreatic cancer. The expression of epithelial and mesenchymal markers was examined by real-time PCR and immunofluorescent staining. The migration of pancreatic cancer cells was examined by scratch and two-chamber assays. Pancreatic cancer cells co-cultured with PSCs showed loose cell contacts and a scattered, fibroblast-like appearance. The expression of E-cadherin, cytokeratin 19, and membrane-associated β-catenin was decreased, whereas vimentin and Snail (Snai-1) expression was increased more in cancer cells co-cultured with PSCs than in mono-cultured cells. The migration of pancreatic cancer cells was increased by co-culture with PSCs. The PSC-induced decrease of E-cadherin expression was not altered by treatment with anti

The physiology of rodent beta-cells in pancreas slices.

Science.gov (United States)

Rupnik, M

2009-01-01

Beta-cells in pancreatic islets form complex syncytia. Sufficient cell-to-cell electrical coupling seems to ensure coordinated depolarization pattern and insulin release that can be further modulated by rich innervation. The complex structure and coordinated action develop after birth during fast proliferation of the endocrine tissue. These emergent properties can be lost due to various reasons later in life and can lead to glucose intolerance and diabetes mellitus. Pancreas slice is a novel method of choice to study the physiology of beta-cells still embedded in their normal cellulo-social context. I present major advantages, list drawbacks and provide an overview on recent advances in our understanding of the physiology of beta-cells using the pancreas slice approach.

Protein-Mediated Interactions of Pancreatic Islet Cells

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Paolo Meda

2013-01-01

Full Text Available The islets of Langerhans collectively form the endocrine pancreas, the organ that is soley responsible for insulin secretion in mammals, and which plays a prominent role in the control of circulating glucose and metabolism. Normal function of these islets implies the coordination of different types of endocrine cells, noticeably of the beta cells which produce insulin. Given that an appropriate secretion of this hormone is vital to the organism, a number of mechanisms have been selected during evolution, which now converge to coordinate beta cell functions. Among these, several mechanisms depend on different families of integral membrane proteins, which ensure direct (cadherins, N-CAM, occludin, and claudins and paracrine communications (pannexins between beta cells, and between these cells and the other islet cell types. Also, other proteins (integrins provide communication of the different islet cell types with the materials that form the islet basal laminae and extracellular matrix. Here, we review what is known about these proteins and their signaling in pancreatic β-cells, with particular emphasis on the signaling provided by Cx36, given that this is the integral membrane protein involved in cell-to-cell communication, which has so far been mostly investigated for effects on beta cell functions.

Cdk5 inhibitory peptide (CIP inhibits Cdk5/p25 activity induced by high glucose in pancreatic beta cells and recovers insulin secretion from p25 damage.

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Ya-Li Zheng

Full Text Available Cdk5/p25 hyperactivity has been demonstrated to lead to neuron apoptosis and degenerations. Chronic exposure to high glucose (HG results in hyperactivity of Cdk5 and reduced insulin secretion. Here, we set out to determine whether abnormal upregulation of Cdk5/p25 activity may be induced in a pancreatic beta cell line, Min6 cells. We first confirmed that p25 were induced in overexpressed p35 cells treated with HG and increased time course dependence. Next, we showed that no p25 was detected under short time HG stimulation (4-12 hrs, however was detectable in the long exposure in HG cells (24 hrs and 48 hrs. Cdk5 activity in the above cells was much higher than low glucose treated cells and resulted in more than 50% inhibition of insulin secretion. We confirmed these results by overexpression of p25 in Min6 cells. As in cortical neurons, CIP, a small peptide, inhibited Cdk5/p25 activity and restored insulin secretion. The same results were detected in co-infection of dominant negative Cdk5 (DNCdk5 with p25. CIP also reduced beta cells apoptosis induced by Cdk5/p25. These studies indicate that Cdk5/p25 hyperactivation deregulates insulin secretion and induces cell death in pancreatic beta cells and suggests that CIP may serve as a therapeutic agent for type 2 diabetes.

Knowledge Gaps in Rodent Pancreas Biology: Taking Human Pluripotent Stem Cell-Derived Pancreatic Beta Cells into Our Own Hands.

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Santosa, Munirah Mohamad; Low, Blaise Su Jun; Pek, Nicole Min Qian; Teo, Adrian Kee Keong

2015-01-01

In the field of stem cell biology and diabetes, we and others seek to derive mature and functional human pancreatic β cells for disease modeling and cell replacement therapy. Traditionally, knowledge gathered from rodents is extended to human pancreas developmental biology research involving human pluripotent stem cells (hPSCs). While much has been learnt from rodent pancreas biology in the early steps toward Pdx1(+) pancreatic progenitors, much less is known about the transition toward Ngn3(+) pancreatic endocrine progenitors. Essentially, the later steps of pancreatic β cell development and maturation remain elusive to date. As a result, the most recent advances in the stem cell and diabetes field have relied upon combinatorial testing of numerous growth factors and chemical compounds in an arbitrary trial-and-error fashion to derive mature and functional human pancreatic β cells from hPSCs. Although this hit-or-miss approach appears to have made some headway in maturing human pancreatic β cells in vitro, its underlying biology is vaguely understood. Therefore, in this mini-review, we discuss some of these late-stage signaling pathways that are involved in human pancreatic β cell differentiation and highlight our current understanding of their relevance in rodent pancreas biology. Our efforts here unravel several novel signaling pathways that can be further studied to shed light on unexplored aspects of rodent pancreas biology. New investigations into these signaling pathways are expected to advance our knowledge in human pancreas developmental biology and to aid in the translation of stem cell biology in the context of diabetes treatments.

β-Cell regeneration through the transdifferentiation of pancreatic cells: Pancreatic progenitor cells in the pancreas.

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Kim, Hyo-Sup; Lee, Moon-Kyu

2016-05-01

Pancreatic progenitor cell research has been in the spotlight, as these cells have the potential to replace pancreatic β-cells for the treatment of type 1 and 2 diabetic patients with the absence or reduction of pancreatic β-cells. During the past few decades, the successful treatment of diabetes through transplantation of the whole pancreas or isolated islets has nearly been achieved. However, novel sources of pancreatic islets or insulin-producing cells are required to provide sufficient amounts of donor tissues. To overcome this limitation, the use of pancreatic progenitor cells is gaining more attention. In particular, pancreatic exocrine cells, such as duct epithelial cells and acinar cells, are attractive candidates for β-cell regeneration because of their differentiation potential and pancreatic lineage characteristics. It has been assumed that β-cell neogenesis from pancreatic progenitor cells could occur in pancreatic ducts in the postnatal stage. Several studies have shown that insulin-producing cells can arise in the duct tissue of the adult pancreas. Acinar cells also might have the potential to differentiate into insulin-producing cells. The present review summarizes recent progress in research on the transdifferentiation of pancreatic exocrine cells into insulin-producing cells, especially duct and acinar cells.

Glucose activates prenyltransferases in pancreatic islet {beta}-cells

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Goalstone, Marc [Department of Medicine, University of Colorado, VA Medical Center, Denver, CO 80220 (United States); Kamath, Vasudeva [Department of Pharmaceutical Sciences, Wayne State University, VA Medical Center, Detroit, MI 48201 (United States); Kowluru, Anjaneyulu, E-mail: akowluru@med.wayne.edu [Department of Pharmaceutical Sciences, Wayne State University, VA Medical Center, Detroit, MI 48201 (United States)

2010-01-01

A growing body of evidence implicates small G-proteins [e.g., Cdc42 and Rac1] in glucose-stimulated insulin secretion [GSIS] in the islet {beta}-cell. These signaling proteins undergo post-translational modifications [e.g., prenylation] at their C-terminal cysteine residue and appear to be essential for the transport and fusion of insulin-containing secretory granules with the plasma membrane and the exocytotic secretion of insulin. However, potential regulation of the prenylating enzymes by physiological insulin secretogues [e.g., glucose] has not been investigated thus far. Herein, we report immunological localization, sub-cellular distribution and regulation of farnesyltransferases [FTases] and geranylgeranyltransferase [GGTase] by glucose in insulin-secreting INS 832/13 {beta}-cells and normal rat islets. Our findings suggest that an insulinotropic concentration of glucose [20 mM] markedly stimulated the expression of the {alpha}-subunits of FTase/GGTase-1, but not the {beta}-subunits of FTase or GGTase-1 without significantly affecting the predominantly cytosolic distribution of these holoenzymes in INS 832/13 cells and rodent islets. Under these conditions, glucose significantly stimulated [2.5- to 4.0-fold over basal] the activities of both FTase and GGTase-1 in both cell types. Together, these findings provide the first evidence to suggest that GSIS involves activation of the endogenous islet prenyltransferases by glucose, culminating in the activation of their respective G-protein substrates, which is necessary for cytoskeletal rearrangement, vesicular transport, fusion and secretion of insulin.

Hypoglycemic and beta cell protective effects of andrographolide analogue for diabetes treatment

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Larrick James W

2009-07-01

Full Text Available Abstract Background While all anti-diabetic agents can decrease blood glucose level directly or indirectly, few are able to protect and preserve both pancreatic beta cell mass and their insulin-secreting functions. Thus, there is an urgent need to find an agent or combination of agents that can lower blood glucose and preserve pancreatic beta cells at the same time. Herein, we report a dual-functional andrographolide-lipoic acid conjugate (AL-1. The anti-diabetic and beta cell protective activities of this novel andrographolide-lipoic acid conjugate were investigated. Methods In alloxan-treated mice (a model of type 1 diabetes, drugs were administered orally once daily for 6 days post-alloxan treatment. Fasting blood glucose and serum insulin were determined. Pathologic and immunohistochemical analysis of pancreatic islets were performed. Translocation of glucose transporter subtype 4 in soleus muscle was detected by western blot. In RIN-m cells in vitro, the effect of AL-1 on H2O2-induced damage and reactive oxidative species production stimulated by high glucose and glibenclamide were measured. Inhibition of nuclear factor kappa B (NF-κB activation induced by IL-1β and IFN-γ was investigated. Results In alloxan-induced diabetic mouse model, AL-1 lowered blood glucose, increased insulin and prevented loss of beta cells and their dysfunction, stimulated glucose transport protein subtype 4 (GLUT4 membrane translocation in soleus muscles. Pretreatment of RIN-m cells with AL-1 prevented H2O2-induced cellular damage, quenched glucose and glibenclamide-stimulated reactive oxidative species production, and inhibited cytokine-stimulated NF-κB activation. Conclusion We have demonstrated that AL-1 had both hypoglycemic and beta cell protective effects which translated into antioxidant and NF-κB inhibitory activity. AL-1 is a potential new anti-diabetic agent.

Dynamics and Synchrony of Pancreatic beta-cells and Islets

DEFF Research Database (Denmark)

Pedersen, Morten Gram

2006-01-01

description of these processes and their interactions would provide important input in the search for a better treatment of the disease. The thesis describes several aspects of mathematical modeling of beta-cells relevant for the understanding of glucose stimulated insulin secretion. It consists...... and the synchronized behavior of many coupled beta-cells as well as to the synchrony of islets. Rather than developing new biophysical models, the thesis investigates existing models, their integration and simplifications, and analyzed the corresponding dynamics, in order to use these models for investigating...

Imaging of beta-Cell Mass and Insulitis in Insulin-Dependent (Type 1) Diabetes Mellitus

NARCIS (Netherlands)

Di Gialleonardo, Valentina; de Vries, Erik F. J.; Di Girolamo, Marco; Quintero, Ana M.; Dierckx, Rudi A. J. O.; Signore, Alberto

2012-01-01

Insulin-dependent (type 1) diabetes mellitus is a metabolic disease with a complex multifactorial etiology and a poorly understood pathogenesis. Genetic and environmental factors cause an autoimmune reaction against pancreatic beta-cells, called insulitis, confirmed in pancreatic samples obtained at

Profile of blood glucose and ultrastucture of beta cells pancreatic islet in alloxan compound induced rats

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I Nyoman Suarsana

2010-06-01

Full Text Available Diabetes is marked by elevated levels of blood glucose, and progressive changes of the structure of pancreatic islet histopathology. The objective of this research was to analyse the glucose level and histophatological feature in pancreatic islet in alloxan compound induced rats. A total of ten male Spraque Dawley rats of 2 months old were used in this study. The rats were divided into two groups: (1 negative control group (K-, and (2 positif induced alloxan group (diabetic group =DM. The rats were induced by a single dose intraperitonial injection of alloxan compound 120 mg/kg of body weight. The treatment was conducted for 28 days. Blood glucose levels of rats were analysed at 0, 4, 7, 14, 21, and 28 days following treatment. At the end of the experiment, rats were sacrificed by cervical dislocation. Pancreas was collected for analysis of histopathological study by Immunohistochemical technique, and ultrastructural study using transmission electron microscope (TEM. The result showed that Langerhans islet of diabetic rat (rat of DM group showed a marked reduction of size, number of Langerhans islet of diabetic rat decrease, and characterized by hyperglycemic condition. By using TEM, beta cells of DM group showed the rupture of mitochondrial membrane, the lost of cisternal structure of inner membrane of mitocondria, reduction of insulin secretory granules, linkage between cells acinar with free Langerhans islet, and the caryopicnotic of nucleus.

Alterations in integrin expression modulates invasion of pancreatic cancer cells.

LENUS (Irish Health Repository)

Walsh, Naomi

2009-01-01

BACKGROUND: Factors mediating the invasion of pancreatic cancer cells through the extracellular matrix (ECM) are not fully understood. METHODS: In this study, sub-populations of the human pancreatic cancer cell line, MiaPaCa-2 were established which displayed differences in invasion, adhesion, anoikis, anchorage-independent growth and integrin expression. RESULTS: Clone #3 displayed higher invasion with less adhesion, while Clone #8 was less invasive with increased adhesion to ECM proteins compared to MiaPaCa-2. Clone #8 was more sensitive to anoikis than Clone #3 and MiaPaCa-2, and displayed low colony-forming efficiency in an anchorage-independent growth assay. Integrins beta 1, alpha 5 and alpha 6 were over-expressed in Clone #8. Using small interfering RNA (siRNA), integrin beta1 knockdown in Clone #8 cells increased invasion through matrigel and fibronectin, increased motility, decreased adhesion and anoikis. Integrin alpha 5 and alpha 6 knockdown also resulted in increased motility, invasion through matrigel and decreased adhesion. CONCLUSION: Our results suggest that altered expression of integrins interacting with different extracellular matrixes may play a significant role in suppressing the aggressive invasive phenotype. Analysis of these clonal populations of MiaPaCa-2 provides a model for investigations into the invasive properties of pancreatic carcinoma.

[Associations of insulin resistance and pancreatic beta-cell function with plasma glucose level in type 2 diabetes].

Science.gov (United States)

Nian, Xiaoping; Sun, Gaisheng; Dou, Chunmei; Hou, Hongbo; Fan, Xiuping; Yu, Hongmei; Ma, Ling; He, Bingxian

2002-06-10

To investigate the influence of insulin resistance and pancreatic beta-cell function on plasma glucose level in type 2 diabetes so as to provide theoretical basis for reasonable selection of hypoglycemic agents. The plasma non-specific insulin (NSINS), true insulin (TI) and glucose in eight-one type 2 diabetics, 38 males and 43 females, with a mean age of 53 years, were examined 0, 30, 60 and 120 minutes after they had 75 grams of instant noodles. The patients were divided into two groups according to their fasting plasma glucose (FPG): group A (FPG = 8.89 mmol/L). The insulin resistance was evaluated by HOMA-IR, the beta-cell function was evaluated by HOMA-beta formula and the formula deltaI(30)/deltaG(30) = (deltaI(30)-deltaI(0))/(deltaG(30)-deltaG(0)). The insulin area under curve (INSAUC) was evaluated by the formula INSAUC=FINS/2+INS(30)+INS(60)+INS(120)/2. The mean FPG was 6.23 mmol/L in group A and 12.6 mmol/L in group B. PG2H was 11.7 mmol/L in group A and 19.2 mmol/L in group B. The TI levels in group B at 0, 30, 60, 120 min during standard meal test were significantly higher than those in group A: 6.15 +/- 1.06 vs 4.77 +/- 1.06, 9.76 +/- 1.1 vs 5.88 +/- 1.1,14.68 +/- 1.11 vs 6.87 +/- 1.1 and 17.13 +/- 1.12 vs 8.0 +/- 1.1 microU/dl (all P< 0.01). The NSINS showed the same trend. The insulin resistance in group B was 1.5 times that in group A. With the insulin resistance adjusted, the beta cell function in group A was 5 to 6 times that in group B. The INSAUC in group A was 1.66 times larger than that in group B, especially the INSAUC for true insulin (2 times larger). The contribution of insulin resistance and beta cell function to PG2H was half by half in group A and 1:8 in group B. beta cell function calculated by insulin (Homa-beta) explained 41% of the plasma glucose changes in group A and 54% of the plasma glucose changes in group B. The contribution of insulin deficiency to plasma glocose was 3.3.times that of insulin resistance in group A and was 9

Visualizing pancreatic {beta}-cell mass with [{sup 11}C]DTBZ

Energy Technology Data Exchange (ETDEWEB)

Simpson, Norman Ray [Department of Radiology, Columbia University Medical School, New York, NY 10032 (United States); Souza, Fabiola [Department of Surgery, Columbia University Medical School, New York, NY 10032 (United States); Witkowski, Piotr [Department of Medicine, Columbia University Medical School, New York, NY 10032 (United States); Maffei, Antonella [Institute of Genetics and Biophysics ' Adriano Buzzati-Traverso' , CNR, Naples 80131 (Italy); Raffo, Anthony [Department of Surgery, Columbia University Medical School, New York, NY 10032 (United States); Herron, Alan [Center for Comparative Medicine and The Department of Pathology, Baylor College of Medicine, Houston, TX 77030 (United States); Kilbourn, Michael [Department of Radiology, University of Michigan, Ann Arbor, MI 48109-0638 (United States); Jurewicz, Agata [Department of Radiology, Columbia University Medical School, New York, NY 10032 (United States); Herold, Kevan [Department of Surgery, Columbia University Medical School, New York, NY 10032 (United States); Liu, Eric [Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, MD 20854 (United States); Hardy, Mark Adam [Department of Medicine, Columbia University Medical School, New York, NY 10032 (United States); Van Heertum, Ronald [Department of Radiology, Columbia University Medical School, New York, NY 10032 (United States); Harris, Paul Emerson [Department of Surgery, Columbia University Medical School, New York, NY 10032 (United States)]. E-mail: peh1@columbia.edu

2006-10-15

{beta}-Cell mass (BCM) influences the total amount of insulin secreted, varies by individual and by the degree of insulin resistance, and is affected by physiologic and pathologic conditions. The islets of Langerhans, however, appear to have a reserve capacity of insulin secretion and, overall, assessments of insulin and blood glucose levels remain poor measures of BCM, {beta}-cell function and progression of diabetes. Thus, novel noninvasive determinations of BCM are needed to provide a quantitative endpoint for novel therapies of diabetes, islet regeneration and transplantation. Built on previous gene expression studies, we tested the hypothesis that the targeting of vesicular monoamine transporter 2 (VMAT2), which is expressed by {beta} cells, with [{sup 11}C]dihydrotetrabenazine ([{sup 11}C]DTBZ), a radioligand specific for VMAT2, and the use of positron emission tomography (PET) can provide a measure of BCM. In this report, we demonstrate decreased radioligand uptake within the pancreas of Lewis rats with streptozotocin-induced diabetes relative to their euglycemic historical controls. These studies suggest that quantitation of VMAT2 expression in {beta} cells with the use of [{sup 11}C]DTBZ and PET represents a method for noninvasive longitudinal estimates of changes in BCM that may be useful in the study and treatment of diabetes.

TGF-{beta}1 increases invasiveness of SW1990 cells through Rac1/ROS/NF-{kappa}B/IL-6/MMP-2

Energy Technology Data Exchange (ETDEWEB)

Binker, Marcelo G. [Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8 (Canada); CBRHC Research Center, Buenos Aires (Argentina); Binker-Cosen, Andres A. [CBRHC Research Center, Buenos Aires (Argentina); Gaisano, Herbert Y. [Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8 (Canada); Cosen, Rodica H. de [CBRHC Research Center, Buenos Aires (Argentina); Cosen-Binker, Laura I., E-mail: laura.cosen.binker@utoronto.ca [Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8 (Canada); CBRHC Research Center, Buenos Aires (Argentina)

2011-02-04

Research highlights: {yields} Rac1 mediates TGF-{beta}1-induced SW1990 invasion through MMP-2 secretion and activation. {yields} NADPH-generated ROS act downstream of Rac1 in TGF-{beta}1-challenged SW1990 cells. {yields} TGF-{beta}1-stimulated ROS activate NF-{kappa}B in SW1990 cells. {yields} NF{kappa}B-induced IL-6 release is required for secretion and activation of MMP-2 in SW1990 cells. -- Abstract: Human pancreatic cancer invasion and metastasis have been found to correlate with increased levels of active matrix metalloproteinase 2 (MMP-2). The multifunctional cytokine transforming growth factor beta 1 (TGF-{beta}1) has been shown to increase both secretion of MMP-2 and invasion by several pancreatic cancer cell types. In the present study, we investigated the signaling pathway involved in TGF-{beta}1-promoted MMP-2 secretion and invasion by human pancreatic cancer cells SW1990. Using specific inhibitors, we found that stimulation of these tumor cells with TGF-{beta}1 induced secretion and activation of the collagenase MMP-2, which was required for TGF-{beta}1-stimulated invasion. Our results also indicate that signaling events involved in TGF-{beta}1-enhanced SW1990 invasiveness comprehend activation of Rac1 followed by generation of reactive oxygen species through nicotinamide adenine dinucleotide phosphate-oxidase, activation of nuclear factor-kappa beta, release of interleukin-6, and secretion and activation of MMP-2.

Repeated Gene Transfection Impairs the Engraftment of Transplanted Porcine Neonatal Pancreatic Cells

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Min Koo Seo

2011-02-01

Full Text Available BackgroundPreviously, we reported that neonatal porcine pancreatic cells transfected with hepatocyte growth factor (HGF gene in an Epstein-Barr virus (EBV-based plasmid (pEBVHGF showed improved proliferation and differentiation compared to those of the control. In this study, we examined if pancreatic cells transfected repeatedly with pEBVHGF can be successfully grafted to control blood glucose in a diabetes mouse model.MethodsNeonatal porcine pancreatic cells were cultured as a monolayer and were transfected with pEBVHGF every other day for a total of three transfections. The transfected pancreatic cells were re-aggregated and transplanted into kidney capsules of diabetic nude mice or normal nude mice. Blood glucose level and body weight were measured every other day after transplantation. The engraftment of the transplanted cells and differentiation into beta cells were assessed using immunohistochemistry.ResultsRe-aggregation of the pancreatic cells before transplantation improved engraftment of the cells and facilitated neovascularization of the graft. Right before transplantation, pancreatic cells that were transfected with pEBVHGF and then re-aggregated showed ductal cell marker expression. However, ductal cells disappeared and the cells underwent fibrosis in a diabetes mouse model two to five weeks after transplantation; these mice also did not show controlled blood glucose levels. Furthermore, pancreatic cells transplanted into nude mice with normal blood glucose showed poor graft survival regardless of the type of transfected plasmid (pCEP4, pHGF, or pEBVHGF.ConclusionFor clinical application of transfected neonatal porcine pancreatic cells, further studies are required to develop methods of overcoming the damage for the cells caused by repeated transfection and to re-aggregate them into islet-like structures.

Calcium has a permissive role in interleukin-1beta-induced c-jun N-terminal kinase activation in insulin-secreting cells

DEFF Research Database (Denmark)

Størling, Joachim; Zaitsev, Sergei V; Kapelioukh, Iouri L

2005-01-01

The c-jun N-terminal kinase (JNK) signaling pathway mediates IL-1beta-induced apoptosis in insulin-secreting cells, a mechanism relevant to the destruction of pancreatic beta-cells in type 1 and 2 diabetes. However, the mechanisms that contribute to IL-1beta activation of JNK in beta-cells are la...

A Simple Matter of Life and Death—The Trials of Postnatal Beta-Cell Mass Regulation

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Elena Tarabra

2012-01-01

Full Text Available Pancreatic beta-cells, which secrete the hormone insulin, are the key arbiters of glucose homeostasis. Defective beta-cell numbers and/or function underlie essentially all major forms of diabetes and must be restored if diabetes is to be cured. Thus, the identification of the molecular regulators of beta-cell mass and a better understanding of the processes of beta-cell differentiation and proliferation may provide further insight for the development of new therapeutic targets for diabetes. This review will focus on the principal hormones and nutrients, as well as downstream signalling pathways regulating beta-cell mass in the adult. Furthermore, we will also address more recently appreciated regulators of beta-cell mass, such as microRNAs.

Response gene to complement-32 enhances metastatic phenotype by mediating transforming growth factor beta-induced epithelial-mesenchymal transition in human pancreatic cancer cell line BxPC-3

Directory of Open Access Journals (Sweden)

Zhu Liang

2012-03-01

Full Text Available Abstract Background Response gene to complement-32 (RGC-32 is comprehensively expressed in many kinds of tissues and has been reported to be expressed abnormally in different kinds of human tumors. However, the role of RGC-32 in cancer remains controversial and no reports have described the effect of RGC-32 in pancreatic cancer. The present study investigated the expression of RGC-32 in pancreatic cancer tissues and explored the role of RGC-32 in transforming growth factor-beta (TGF-β-induced epithelial-mesenchymal transition (EMT in human pancreatic cancer cell line BxPC-3. Methods Immunohistochemical staining of RGC-32 and E-cadherin was performed on specimens from 42 patients with pancreatic cancer, 12 with chronic pancreatitis and 8 with normal pancreas. To evaluate the role of RGC-32 in TGF-β-induced EMT in pancreatic cancer cells, BxPC-3 cells were treated with TGF-β1, and RGC-32 siRNA silencing and gene overexpression were performed as well. The mRNA expression and protein expression of RGC-32 and EMT markers such E-cadherin and vimentin were determined by quantitative reverse transcription-PCR (qRT-PCR and western blot respectively. Finally, migration ability of BxPC-3 cells treated with TGF-β and RGC-32 siRNA transfection was examined by transwell cell migration assay. Results We found stronger expression of RGC-32 and higher abnormal expression rate of E-cadherin in pancreatic cancer tissues than those in chronic pancreatitis tissues and normal pancreatic tissues. Immunohistochemical analysis revealed that both RGC-32 positive expression and E-cadherin abnormal expression in pancreatic cancer were correlated with lymph node metastasis and TNM staging. In addition, a significant and positive correlation was found between positive expression of RGC-32 and abnormal expression of E-cadherin. Furthermore, in vitro, we found sustained TGF-β stimuli induced EMT and up-regulated RGC-32 expression in BxPC-3 cells. By means of si

Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells.

Science.gov (United States)

Rostovskaya, Maria; Bredenkamp, Nicholas; Smith, Austin

2015-10-19

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification. © 2015 The Authors.

Alkali pH directly activates ATP-sensitive K+ channels and inhibits insulin secretion in beta-cells.

Science.gov (United States)

Manning Fox, Jocelyn E; Karaman, Gunce; Wheeler, Michael B

2006-11-17

Glucose stimulation of pancreatic beta-cells is reported to lead to sustained alkalization, while extracellular application of weak bases is reported to inhibit electrical activity and decrease insulin secretion. We hypothesize that beta-cell K(ATP) channel activity is modulated by alkaline pH. Using the excised patch-clamp technique, we demonstrate a direct stimulatory action of alkali pH on recombinant SUR1/Kir6.2 channels due to increased open probability. Bath application of alkali pH similarly activates native islet beta-cell K(ATP) channels, leading to an inhibition of action potentials, and hyperpolarization of membrane potential. In situ pancreatic perfusion confirms that these cellular effects of alkali pH are observable at a functional level, resulting in decreases in both phase 1 and phase 2 glucose-stimulated insulin secretion. Our data are the first to report a stimulatory effect of a range of alkali pH on K(ATP) channel activity and link this to downstream effects on islet beta-cell function.

Suppressor of cytokine signalling (SOCS)-3 protects beta cells against IL-1beta-mediated toxicity through inhibition of multiple nuclear factor-kappaB-regulated proapoptotic pathways

DEFF Research Database (Denmark)

Karlsen, Allan Ertman; Heding, P E; Frobøse, H

2004-01-01

The proinflammatory cytokine IL-1beta induces apoptosis in pancreatic beta cells via pathways dependent on nuclear factor-kappaB (NF-kappaB), mitogen-activated protein kinase, and protein kinase C. We recently showed suppressor of cytokine signalling (SOCS)-3 to be a natural negative feedback reg...... regulator of IL-1beta- and IFN-gamma-mediated signalling in rat islets and beta cell lines, preventing their deleterious effects. However, the mechanisms underlying SOCS-3 inhibition of IL-1beta signalling and prevention against apoptosis remain unknown....

Chemistry and biology of reactive species with special reference to the antioxidative defence status in pancreatic β-cells.

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Lenzen, Sigurd

2017-08-01

Diabetes mellitus is a serious metabolic disease. Dysfunction and subsequent loss of the β-cells in the islets of Langerhans through apoptosis ultimately cause a life-threatening insulin deficiency. The underlying reason for the particular vulnerability of the β-cells is an extraordinary sensitivity to the toxicity of reactive oxygen and nitrogen species (ROS and RNS) due to its low antioxidative defense status. This review considers the different aspects of the chemistry and biology of the biologically most important reactive species and their chemico-biological interactions in the β-cell toxicity of proinflammatory cytokines in type 1 diabetes and of lipotoxicity in type 2 diabetes development. The weak antioxidative defense equipment in the different subcellular organelles makes the β-cells particularly vulnerable and prone to mitochondrial, peroxisomal and ER stress. Looking upon the enzyme deficiencies which are responsible for the low antioxidative defense status of the pancreatic β-cells it is the lack of enzymatic capacity for H 2 O 2 inactivation at all major subcellular sites. Diabetes is the most prevalent metabolic disorder with a steadily increasing incidence of both type 1 and type 2 diabetes worldwide. The weak protection of the pancreatic β-cells against oxidative stress is a major reason for their particular vulnerability. Thus, careful protection of the β-cells is required for prevention of the disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Reprogramming of various cell types to a beta-like state by Pdx1, Ngn3 and MafA.

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Ersin Akinci

Full Text Available The three transcription factors, PDX1, NGN3 and MAFA, are very important in pancreatic development. Overexpression of these three factors can reprogram both pancreatic exocrine cells and SOX9-positive cells of the liver into cells resembling pancreatic beta cells. In this study we investigate whether other cell types can be reprogrammed. Eight cell types are compared and the results are consistent with the idea that reprogramming occurs to a greater degree for developmentally related cells (pancreas, liver than for other types, such as fibroblasts. Using a line of mouse hepatocyte-derived cells we screened 13 compounds for the ability to increase the yield of reprogrammed cells. Three are active and when used in combination they can increase the yield of insulin-immunopositive cells by a factor of six. These results should contribute to the eventual ability to develop a new cure for diabetes based on the ability to reprogram other cells in the body to a beta cell phenotype.

PDX1, Neurogenin-3, and MAFA: critical transcription regulators for beta cell development and regeneration

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Yaxi Zhu

2017-11-01

Full Text Available Abstract Transcription factors regulate gene expression through binding to specific enhancer sequences. Pancreas/duodenum homeobox protein 1 (PDX1, Neurogenin-3 (NEUROG3, and V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA are transcription factors critical for beta cell development and maturation. NEUROG3 is expressed in endocrine progenitor cells and controls islet differentiation and regeneration. PDX1 is essential for the development of pancreatic exocrine and endocrine cells including beta cells. PDX1 also binds to the regulatory elements and increases insulin gene transcription. Likewise, MAFA binds to the enhancer/promoter region of the insulin gene and drives insulin expression in response to glucose. In addition to those natural roles in beta cell development and maturation, ectopic expression of PDX1, NEUROG3, and/or MAFA has been successfully used to reprogram various cell types into insulin-producing cells in vitro and in vivo, such as pancreatic exocrine cells, hepatocytes, and pluripotent stem cells. Here, we review biological properties of PDX1, NEUROG3, and MAFA, and their applications and limitations for beta cell regenerative approaches. The primary source literature for this review was acquired using a PubMed search for articles published between 1990 and 2017. Search terms include diabetes, insulin, trans-differentiation, stem cells, and regenerative medicine.

Can pancreatic duct-derived progenitors be a source of islet regeneration?

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Xia, Bing [Department of Endocrinology, First Hospital of Harbin Medical University, Harbin, Hei Long Jiang Province 150001 (China); Zhan, Xiao-Rong, E-mail: xiaorongzhan@sina.com [Department of Endocrinology, First Hospital of Harbin Medical University, Harbin, Hei Long Jiang Province 150001 (China); Yi, Ran [Department of Endocrinology, First Hospital of Harbin Medical University, Harbin, Hei Long Jiang Province 150001 (China); Yang, Baofeng [Department of Pharmacology, State Key Laboratory of Biomedicine and Pharmacology, Harbin Medical University, Harbin, Hei Long Jiang Province 150001 (China)

2009-06-12

The regenerative process of the pancreas is of interest because the main pathogenesis of diabetes mellitus is an inadequate number of insulin-producing {beta}-cells. The functional mass of {beta}-cells is decreased in type 1 diabetes, so replacing missing {beta}-cells or triggering their regeneration may allow for improved type 1 diabetes treatment. Therefore, expansion of the {beta}-cell mass from endogenous sources, either in vivo or in vitro, represents an area of increasing interest. The mechanism of islet regeneration remains poorly understood, but the identification of islet progenitor sources is critical for understanding {beta}-cell regeneration. One potential source is the islet proper, via the dedifferentiation, proliferation, and redifferentiation of facultative progenitors residing within the islet. Neogenesis, or that the new pancreatic islets can derive from progenitor cells present within the ducts has been reported, but the existence and identity of the progenitor cells have been debated. In this review, we focus on pancreatic ductal cells, which are islet progenitors capable of differentiating into islet {beta}-cells. Islet neogenesis, seen as budding of hormone-positive cells from the ductal epithelium, is considered to be one mechanism for normal islet growth after birth and in regeneration, and has suggested the presence of pancreatic stem cells. Numerous results support the neogenesis hypothesis, the evidence for the hypothesis in the adult comes primarily from morphological studies that have in common the production of damage to all or part of the pancreas, with consequent inflammation and repair. Although numerous studies support a ductal origin for new islets after birth, lineage-tracing experiments are considered the 'gold standard' of proof. Lineage-tracing experiments show that pancreatic duct cells act as progenitors, giving rise to new islets after birth and after injury. The identification of differentiated pancreatic ductal

Foodborne cereulide causes beta-cell dysfunction and apoptosis.

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Roman Vangoitsenhoven

Full Text Available To study the effects of cereulide, a food toxin often found at low concentrations in take-away meals, on beta-cell survival and function.Cell death was quantified by Hoechst/Propidium Iodide in mouse (MIN6 and rat (INS-1E beta-cell lines, whole mouse islets and control cell lines (HepG2 and COS-1. Beta-cell function was studied by glucose-stimulated insulin secretion (GSIS. Mechanisms of toxicity were evaluated in MIN6 cells by mRNA profiling, electron microscopy and mitochondrial function tests.24 h exposure to 5 ng/ml cereulide rendered almost all MIN6, INS-1E and pancreatic islets apoptotic, whereas cell death did not increase in the control cell lines. In MIN6 cells and murine islets, GSIS capacity was lost following 24 h exposure to 0.5 ng/ml cereulide (P<0.05. Cereulide exposure induced markers of mitochondrial stress including Puma (p53 up-regulated modulator of apoptosis, P<0.05 and general pro-apoptotic signals as Chop (CCAAT/-enhancer-binding protein homologous protein. Mitochondria appeared swollen upon transmission electron microscopy, basal respiration rate was reduced by 52% (P<0.05 and reactive oxygen species increased by more than twofold (P<0.05 following 24 h exposure to 0.25 and 0.50 ng/ml cereulide, respectively.Cereulide causes apoptotic beta-cell death at low concentrations and impairs beta-cell function at even lower concentrations, with mitochondrial dysfunction underlying these defects. Thus, exposure to cereulide even at concentrations too low to cause systemic effects appears deleterious to the beta-cell.

GTP- and GDP-Dependent Rab27a Effectors in Pancreatic Beta-Cells.

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Yamaoka, Mami; Ishizaki, Toshimasa; Kimura, Toshihide

2015-01-01

Small guanosine triphosphatases (GTPases) participate in a wide variety of cellular functions including proliferation, differentiation, adhesion, and intracellular transport. Conventionally, only the guanosine 5'-triphosphate (GTP)-bound small GTPase interacts with effector proteins, and the resulting downstream signals control specific cellular functions. Therefore, the GTP-bound form is regarded as active, and the focus has been on searching for proteins that bind the GTP form to look for their effectors. The Rab family small GTPase Rab27a is highly expressed in some secretory cells and is involved in the control of membrane traffic. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in pancreatic beta-cells. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytic stages via its GTP-dependent effectors. We previously identified novel guanosine 5'-diphosphate (GDP)-bound Rab27-interacting proteins. Interestingly, GDP-bound Rab27a controls endocytosis of the secretory membrane via its interaction with these proteins. We also demonstrated that the insulin secretagogue glucose converts Rab27a from its GTP- to GDP-bound forms. Thus, GTP- and GDP-bound Rab27a regulate pre-exocytic and endocytic stages in membrane traffic, respectively. Since the physiological importance of GDP-bound GTPases has been largely overlooked, we consider that the investigation of GDP-dependent effectors for other GTPases is necessary for further understanding of cellular function.

Paracrine GABA and insulin regulate pancreatic alpha cell proliferation in a mouse model of type 1 diabetes.

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Feng, Allen L; Xiang, Yun-Yan; Gui, Le; Kaltsidis, Gesthika; Feng, Qingping; Lu, Wei-Yang

2017-06-01

This study aimed to elucidate the mechanism of increased proliferation of alpha cells in recent-onset type 1 diabetes. Pancreatic beta cells express GAD and produce γ-aminobutyric acid (GABA), which inhibits alpha cell secretion of glucagon. We explored the roles of GABA in alpha cell proliferation in conditions corresponding to type 1 diabetes in a mouse model and in vitro. Type 1 diabetes was induced by injecting the mice with streptozotocin (STZ). Some of the STZ-injected mice were treated with GABA (10 mg/kg daily) for 12 days. Isolated pancreatic islets were treated with STZ or STZ together with GABA for 2 days. The effects of GABA treatment on STZ-induced alpha cell proliferation in vivo and in vitro were assessed. The effect of muscimol, a GABA receptor agonist, on αTC1-6 cell proliferation was also examined. STZ injection substantially decreased levels of GAD, GABA and insulin in pancreatic beta cells 12 h after injection; this was followed by an upsurge of phosphorylated mechanistic target of rapamycin (p-mTOR) in the alpha cells at day 1, and a significant increase in alpha cell mass at day 3. Treating STZ-injected mice with GABA largely restored the immunodetectable levels of insulin and GAD in the beta cells and significantly decreased the number of aldehyde dehydrogenase 1 family, member A3 (ALDH1a3)-positive cells, alpha cell mass and hyperglucagonaemia. STZ treatment also increased alpha cell proliferation in isolated islets, which was reversed by co-treatment with GABA. Muscimol, together with insulin, significantly lowered the level of cytosolic Ca 2+ and p-mTOR, and decreased the proliferation rate of αTC1-6 cells. GABA signalling critically controls the alpha cell population in pancreatic islets. Low intraislet GABA may contribute to alpha cell hyperplasia in early type 1 diabetes.

Antibody Response to Serpin B13 Induces Adaptive Changes in Mouse Pancreatic Islets and Slows Down the Decline in the Residual Beta Cell Function in Children with Recent Onset of Type 1 Diabetes Mellitus.

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Kryvalap, Yury; Lo, Chi-Wen; Manuylova, Ekaterina; Baldzizhar, Raman; Jospe, Nicholas; Czyzyk, Jan

2016-01-01

Type 1 diabetes mellitus (T1D) is characterized by a heightened antibody (Ab) response to pancreatic islet self-antigens, which is a biomarker of progressive islet pathology. We recently identified a novel antibody to clade B serpin that reduces islet-associated T cell accumulation and is linked to the delayed onset of T1D. As natural immunity to clade B arises early in life, we hypothesized that it may influence islet development during that time. To test this possibility healthy young Balb/c male mice were injected with serpin B13 mAb or IgG control and examined for the number and cellularity of pancreatic islets by immunofluorescence and FACS. Beta cell proliferation was assessed by measuring nucleotide analog 5-ethynyl-2'-deoxyuridine (5-EdU) incorporation into the DNA and islet Reg gene expression was measured by real time PCR. Human studies involved measuring anti-serpin B13 autoantibodies by Luminex. We found that injecting anti-serpin B13 monoclonal Ab enhanced beta cell proliferation and Reg gene expression, induced the generation of ∼80 pancreatic islets per animal, and ultimately led to increase in the beta cell mass. These findings are relevant to human T1D because our analysis of subjects just diagnosed with T1D revealed an association between baseline anti-serpin activity and slower residual beta cell function decline in the first year after the onset of diabetes. Our findings reveal a new role for the anti-serpin immunological response in promoting adaptive changes in the endocrine pancreas and suggests that enhancement of this response could potentially help impede the progression of T1D in humans. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Co-culture of neural crest stem cells (NCSC and insulin producing beta-TC6 cells results in cadherin junctions and protection against cytokine-induced beta-cell death.

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Anongnad Ngamjariyawat

Full Text Available PURPOSE: Transplantation of pancreatic islets to Type 1 diabetes patients is hampered by inflammatory reactions at the transplantation site leading to dysfunction and death of insulin producing beta-cells. Recently we have shown that co-transplantation of neural crest stem cells (NCSCs together with the islet cells improves transplantation outcome. The aim of the present investigation was to describe in vitro interactions between NCSCs and insulin producing beta-TC6 cells that may mediate protection against cytokine-induced beta-cell death. PROCEDURES: Beta-TC6 and NCSC cells were cultured either alone or together, and either with or without cell culture inserts. The cultures were then exposed to the pro-inflammatory cytokines IL-1β and IFN-γ for 48 hours followed by analysis of cell death rates (flow cytometry, nitrite production (Griess reagent, protein localization (immunofluorescence and protein phosphorylation (flow cytometry. RESULTS: We observed that beta-TC6 cells co-cultured with NCSCs were protected against cytokine-induced cell death, but not when separated by cell culture inserts. This occurred in parallel with (i augmented production of nitrite from beta-TC6 cells, indicating that increased cell survival allows a sustained production of nitric oxide; (ii NCSC-derived laminin production; (iii decreased phospho-FAK staining in beta-TC6 cell focal adhesions, and (iv decreased beta-TC6 cell phosphorylation of ERK(T202/Y204, FAK(Y397 and FAK(Y576. Furthermore, co-culture also resulted in cadherin and beta-catenin accumulations at the NCSC/beta-TC6 cell junctions. Finally, the gap junction inhibitor carbenoxolone did not affect cytokine-induced beta-cell death during co-culture with NCSCs. CONCLUSION: In summary, direct contacts, but not soluble factors, promote improved beta-TC6 viability when co-cultured with NCSCs. We hypothesize that cadherin junctions between NCSC and beta-TC6 cells promote powerful signals that maintain beta-cell

Diabetes mellitus in Tropical Chronic Pancreatitis Is Not Just a Secondary Type of Diabetes

OpenAIRE

Rossi, L.; Parvin, S.; Hassan, Z.; Hildebrand, P.; Keller, U.; Ali, L.; Beglinger, C.; Azad Khan, A. K.; Whitcomb, David C.; Gyr, N.

2004-01-01

AIMS: In chronic calcific pancreatitis of the tropics, etiology and relationship to developing diabetes mellitus are unknown. Some consider these cases a straightforward secondary type of diabetes, while others suggest selective beta-cell impairment. Testing pancreatic function, we investigated whether selective beta-cell impairment triggers diabetes associated with tropical pancreatitis. METHODS: At a Bangladeshi research institute, 8 chronic tropical pancreatitis and no diabetes mellitus su...

The enteroinsular axis and endocrine pancreatic function in chronic alcohol consumers: evidence for early beta-cell hypofunction.

Science.gov (United States)

Patto, R J; Russo, E K; Borges, D R; Neves, M M

1993-09-01

Chronic alcohol consumers may have, as judged by functional criteria, exocrine as well as endocrine pancreatic dysfunction, the latter represented by a decreased insulin response to an oral glucose load. To investigate whether this decreased insulin response was due to an ethanol-induced beta-cell dysfunction or to an ethanol-induced dysfunction of the enteroinsular axis, we determined glucose, insulin, and C-peptide plasma concentrations following an oral and an intravenous glucose load in 16 healthy volunteer nonalcohol consumers and in 10 chronic alcohol consumers. In each group, total integrated response for glucose did not significantly change whether glucose was given orally or intravenously, indicating isoglycemic glucose loads. The total integrated response values for insulin in the alcoholic group following both glucose loads as well as C-peptide plasma concentrations were significantly lower than in the control group. Moreover, in both groups the insulin TIR values following the oral glucose load were significantly greater than the values obtained following the intravenous glucose load, indicating an incretin effect. These results indicate that the decreased insulin response observed in alcoholics was not caused by a dysfunction of the enteroinsular axis because it also occurred following an intravenous glucose load, but by an ethanol-induced beta-cell dysfunction because C-peptide and insulin were proportionally decreased in this group.

Role of MicroRNAs in Islet Beta-Cell Compensation and Failure during Diabetes

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Valérie Plaisance

2014-01-01

Full Text Available Pancreatic beta-cell function and mass are markedly adaptive to compensate for the changes in insulin requirement observed during several situations such as pregnancy, obesity, glucocorticoids excess, or administration. This requires a beta-cell compensation which is achieved through a gain of beta-cell mass and function. Elucidating the physiological mechanisms that promote functional beta-cell mass expansion and that protect cells against death, is a key therapeutic target for diabetes. In this respect, several recent studies have emphasized the instrumental role of microRNAs in the control of beta-cell function. MicroRNAs are negative regulators of gene expression, and are pivotal for the control of beta-cell proliferation, function, and survival. On the one hand, changes in specific microRNA levels have been associated with beta-cell compensation and are triggered by hormones or bioactive peptides that promote beta-cell survival and function. Conversely, modifications in the expression of other specific microRNAs contribute to beta-cell dysfunction and death elicited by diabetogenic factors including, cytokines, chronic hyperlipidemia, hyperglycemia, and oxidized LDL. This review underlines the importance of targeting the microRNA network for future innovative therapies aiming at preventing the beta-cell decline in diabetes.

Exosomes Derived From Pancreatic Stellate Cells: MicroRNA Signature and Effects on Pancreatic Cancer Cells.

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Takikawa, Tetsuya; Masamune, Atsushi; Yoshida, Naoki; Hamada, Shin; Kogure, Takayuki; Shimosegawa, Tooru

2017-01-01

Pancreatic stellate cells (PSCs) interact with pancreatic cancer cells in the tumor microenvironment. Cell constituents including microRNAs may be exported from cells within membranous nanovesicles termed exosomes. Exosomes might play a pivotal role in intercellular communication. This study aimed to clarify the microRNA signature of PSC-derived exosomes and their effects on pancreatic cancer cells. Exosomes were prepared from the conditioned medium of immortalized human PSCs. MicroRNAs were prepared from the exosomes and their source PSCs, and the microRNA expression profiles were compared by microarray. The effects of PSC-derived exosomes on proliferation, migration, and the mRNA expression profiles were examined in pancreatic cancer cells. Pancreatic stellate cell-derived exosomes contained a variety of microRNAs including miR-21-5p. Several microRNAs such as miR-451a were enriched in exosomes compared to their source PSCs. Pancreatic stellate cell-derived exosomes stimulated the proliferation, migration and expression of mRNAs for chemokine (C - X - C motif) ligands 1 and 2 in pancreatic cancer cells. The stimulation of proliferation, migration, and chemokine gene expression by the conditioned medium of PSCs was suppressed by GW4869, an exosome inhibitor. We clarified the microRNA expression profile in PSC-derived exosomes. Pancreatic stellate cell-derived exosomes might play a role in the interactions between PSCs and pancreatic cancer cells.

In vitro modeling of human pancreatic duct epithelial cell transformation defines gene expression changes induced by K-ras oncogenic activation in pancreatic carcinogenesis.

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Qian, Jiaying; Niu, Jiangong; Li, Ming; Chiao, Paul J; Tsao, Ming-Sound

2005-06-15

Genetic analysis of pancreatic ductal adenocarcinomas and their putative precursor lesions, pancreatic intraepithelial neoplasias (PanIN), has shown a multistep molecular paradigm for duct cell carcinogenesis. Mutational activation or inactivation of the K-ras, p16(INK4A), Smad4, and p53 genes occur at progressive and high frequencies in these lesions. Oncogenic activation of the K-ras gene occurs in >90% of pancreatic ductal carcinoma and is found early in the PanIN-carcinoma sequence, but its functional roles remain poorly understood. We show here that the expression of K-ras(G12V) oncogene in a near diploid HPV16-E6E7 gene immortalized human pancreatic duct epithelial cell line originally derived from normal pancreas induced the formation of carcinoma in 50% of severe combined immunodeficient mice implanted with these cells. A tumor cell line established from one of these tumors formed ductal cancer when implanted orthotopically. These cells also showed increased activation of the mitogen-activated protein kinase, AKT, and nuclear factor-kappaB pathways. Microarray expression profiling studies identified 584 genes whose expression seemed specifically up-regulated by the K-ras oncogene expression. Forty-two of these genes have been reported previously as differentially overexpressed in pancreatic cancer cell lines or primary tumors. Real-time PCR confirmed the overexpression of a large number of these genes. Immunohistochemistry done on tissue microarrays constructed from PanIN and pancreatic cancer samples showed laminin beta3 overexpression starting in high-grade PanINs and occurring in >90% of pancreatic ductal carcinoma. The in vitro modeling of human pancreatic duct epithelial cell transformation may provide mechanistic insights on gene expression changes that occur during multistage pancreatic duct cell carcinogenesis.

Selective destruction of mouse islet beta cells by human T lymphocytes in a newly-established humanized type 1 diabetic model

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Zhao, Yong, E-mail: yongzhao@uic.edu [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Guo, Chengshan; Hwang, David; Lin, Brian; Dingeldein, Michael; Mihailescu, Dan; Sam, Susan; Sidhwani, Seema [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Zhang, Yongkang [Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Jain, Sumit [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Skidgel, Randal A. [Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Prabhakar, Bellur S. [Department of Immunology and Microbiology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Mazzone, Theodore [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Holterman, Mark J. [Department of Surgery, University of Illinois at Chicago, Chicago, IL 60612 (United States)

2010-09-03

Research highlights: {yields} Establish a human immune-mediated type 1 diabetic model in NOD-scid IL2r{gamma}{sup null} mice. {yields} Using the irradiated diabetic NOD mouse spleen mononuclear cells as trigger. {yields} The islet {beta} cells were selectively destroyed by infiltrated human T cells. {yields} The model can facilitate translational research to find a cure for type 1 diabetes. -- Abstract: Type 1 diabetes (T1D) is caused by a T cell-mediated autoimmune response that leads to the loss of insulin-producing {beta} cells. The optimal preclinical testing of promising therapies would be aided by a humanized immune-mediated T1D model. We develop this model in NOD-scid IL2r{gamma}{sup null} mice. The selective destruction of pancreatic islet {beta} cells was mediated by human T lymphocytes after an initial trigger was supplied by the injection of irradiated spleen mononuclear cells (SMC) from diabetic nonobese diabetic (NOD) mice. This resulted in severe insulitis, a marked loss of total {beta}-cell mass, and other related phenotypes of T1D. The migration of human T cells to pancreatic islets was controlled by the {beta} cell-produced highly conserved chemokine stromal cell-derived factor 1 (SDF-1) and its receptor C-X-C chemokine receptor (CXCR) 4, as demonstrated by in vivo blocking experiments using antibody to CXCR4. The specificity of humanized T cell-mediated immune responses against islet {beta} cells was generated by the local inflammatory microenvironment in pancreatic islets including human CD4{sup +} T cell infiltration and clonal expansion, and the mouse islet {beta}-cell-derived CD1d-mediated human iNKT activation. The selective destruction of mouse islet {beta} cells by a human T cell-mediated immune response in this humanized T1D model can mimic those observed in T1D patients. This model can provide a valuable tool for translational research into T1D.

Expansion of Adult Human Pancreatic Tissue Yields Organoids Harboring Progenitor Cells with Endocrine Differentiation Potential

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Cindy J.M. Loomans

2018-03-01

Full Text Available Summary: Generating an unlimited source of human insulin-producing cells is a prerequisite to advance β cell replacement therapy for diabetes. Here, we describe a 3D culture system that supports the expansion of adult human pancreatic tissue and the generation of a cell subpopulation with progenitor characteristics. These cells display high aldehyde dehydrogenase activity (ALDHhi, express pancreatic progenitors markers (PDX1, PTF1A, CPA1, and MYC, and can form new organoids in contrast to ALDHlo cells. Interestingly, gene expression profiling revealed that ALDHhi cells are closer to human fetal pancreatic tissue compared with adult pancreatic tissue. Endocrine lineage markers were detected upon in vitro differentiation. Engrafted organoids differentiated toward insulin-positive (INS+ cells, and circulating human C-peptide was detected upon glucose challenge 1 month after transplantation. Engrafted ALDHhi cells formed INS+ cells. We conclude that adult human pancreatic tissue has potential for expansion into 3D structures harboring progenitor cells with endocrine differentiation potential. : In the context of β cell replacement therapy for diabetes, de Koning and colleagues describe a 3D culture platform that supports ex vivo expansion of human pancreatic tissue as organoids. These organoids harbor a subpopulation of ALDHhi cells that display proliferative capacity and can differentiate to an endocrine fate. Keywords: pancreas, organoid, human, ALDH, endocrine differentiation, beta cells, insulin, progenitor, fetal, diabetes

D-saccharic acid-1,4-lactone ameliorates alloxan-induced diabetes mellitus and oxidative stress in rats through inhibiting pancreatic beta-cells from apoptosis via mitochondrial dependent pathway

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Bhattacharya, Semantee [Department of Life Sciences and Biotechnology, Jadavpur University, 188, Raja S C Mullick Road, Kolkata 700 032 (India); Manna, Prasenjit [Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata-700054 (India); Gachhui, Ratan [Department of Life Sciences and Biotechnology, Jadavpur University, 188, Raja S C Mullick Road, Kolkata 700 032 (India); Sil, Parames C., E-mail: parames@bosemain.boseinst.ac.in [Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata-700054 (India)

2011-12-15

Oxidative stress plays a vital role in diabetic complications. To suppress the oxidative stress mediated damage in diabetic pathophysiology, a special focus has been given on naturally occurring antioxidants present in normal diet. D-saccharic acid 1,4-lactone (DSL), a derivative of D-glucaric acid, is present in many dietary plants and is known for its detoxifying and antioxidant properties. The aim of the present study was to evaluate the beneficial role of DSL against alloxan (ALX) induced diabetes in the pancreas tissue of Swiss albino rats. A dose-dependent study for DSL (20-120 mg/kg body weight) was carried out to find the effective dose of the compound in ALX-induced diabetic rats. ALX exposure elevated the blood glucose, glycosylated Hb, decreased the plasma insulin and disturbed the intra-cellular antioxidant machineries whereas oral administration of DSL at a dose of 80 mg/kg body weight restored these alterations close to normal. Investigating the mechanism of the protective activity of DSL we observed that it prevented the pancreatic {beta}-cell apoptosis via mitochondria-dependent pathway. Results showed decreased mitochondrial membrane potential, enhanced cytochrome c release in the cytosol and reciprocal regulation of Bcl-2 family proteins in the diabetic rats. These events were also found to be associated with increased level of Apaf-1, caspase 9, and caspase 3 that ultimately led to pancreatic {beta}-cell apoptosis. DSL treatment, however, counteracted these changes. In conclusion, DSL possesses the capability of ameliorating the oxidative stress in ALX-induced diabetes and thus could be a promising approach in lessening diabetic complications. Highlights: Black-Right-Pointing-Pointer Oxidative stress is suggested as a key event in the pathogenesis of diabetes. Black-Right-Pointing-Pointer D-saccharic acid 1,4-lactone (DSL) reduces the alloxan-induced diabetes mellitus. Black-Right-Pointing-Pointer DSL normalizes cellular antioxidant machineries

Central infusion of leptin improves insulin resistance and suppresses beta-cell function, but not beta-cell mass, primarily through the sympathetic nervous system in a type 2 diabetic rat model.

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Park, Sunmin; Ahn, Il Sung; Kim, Da Sol

2010-06-05

We investigated whether hypothalamic leptin alters beta-cell function and mass directly via the sympathetic nervous system (SNS) or indirectly as the result of altered insulin resistant states. The 90% pancreatectomized male Sprague Dawley rats had sympathectomy into the pancreas by applying phenol into the descending aorta (SNSX) or its sham operation (Sham). Each group was divided into two sections, receiving either leptin at 300ng/kgbw/h or artificial cerebrospinal fluid (aCSF) via intracerebroventricular (ICV) infusion for 3h as a short-term study. After finishing the infusion study, ICV leptin (3mug/kg bw/day) or ICV aCSF (control) was infused in rats fed 30 energy % fat diets by osmotic pump for 4weeks. At the end of the long-term study, glucose-stimulated insulin secretion and islet morphometry were analyzed. Acute ICV leptin administration in Sham rats, but not in SNSX rats, suppressed the first- and second-phase insulin secretion at hyperglycemic clamp by about 48% compared to the control. Regardless of SNSX, the 4-week administration of ICV leptin improved glucose tolerance during oral glucose tolerance tests and insulin sensitivity at hyperglycemic clamp, compared to the control, while it suppressed second-phase insulin secretion in Sham rats but not in SNSX rats. However, the pancreatic beta-cell area and mass were not affected by leptin and SNSX, though ICV leptin decreased individual beta-cell size and concomitantly increased beta-cell apoptosis in Sham rats. Leptin directly decreases insulin secretion capacity mainly through the activation of SNS without modulating pancreatic beta-cell mass.

Hormone-sensitive lipase deficiency suppresses insulin secretion from pancreatic islets of Lepob/ob mice

International Nuclear Information System (INIS)

Sekiya, Motohiro; Yahagi, Naoya; Tamura, Yoshiaki; Okazaki, Hiroaki; Igarashi, Masaki; Ohta, Keisuke; Takanashi, Mikio; Kumagai, Masayoshi; Takase, Satoru; Nishi, Makiko; Takeuchi, Yoshinori; Izumida, Yoshihiko; Kubota, Midori; Ohashi, Ken; Iizuka, Yoko; Yagyu, Hiroaki; Gotoda, Takanari; Nagai, Ryozo; Shimano, Hitoshi; Yamada, Nobuhiro

2009-01-01

It has long been a matter of debate whether the hormone-sensitive lipase (HSL)-mediated lipolysis in pancreatic β-cells can affect insulin secretion through the alteration of lipotoxicity. We generated mice lacking both leptin and HSL (Lep ob/ob /HSL -/- ) and explored the role of HSL in pancreatic β-cells in the setting of obesity. Lep ob/ob /HSL -/- developed elevated blood glucose levels and reduced plasma insulin levels compared with Lep ob/ob /HSL +/+ in a fed state, while the deficiency of HSL did not affect glucose homeostasis in Lep +/+ background. The deficiency of HSL exacerbated the accumulation of triglycerides in Lep ob/ob islets, leading to reduced glucose-stimulated insulin secretion. The deficiency of HSL also diminished the islet mass in Lep ob/ob mice due to decreased cell proliferation. In conclusion, HSL affects insulin secretary capacity especially in the setting of obesity.

Insulin Resistance and Impaired Pancreatic β-Cell Function in Adult Offspring of Women With Diabetes in Pregnancy

DEFF Research Database (Denmark)

Kelstrup, Louise; Damm, Peter; Mathiesen, Elisabeth R

2013-01-01

Context:Offspring of women with diabetes during pregnancy have increased risk of glucose intolerance in adulthood, but the underlying mechanisms are unknown.Objective:We aimed to investigate effects of intrauterine hyperglycemia on insulin secretion and - action in adult offspring of mothers...... a standard oral glucose tolerance test (120 minutes, 75 gram glucose). Pancreatic beta-cell function taking the prevailing insulin sensitivity into account was estimated by disposition indices.Results:Both groups of offspring exposed during pregnancy to either maternal gestational diabetes or type 1 diabetes.......005).Conclusion:Reduced insulin sensitivity as well as impaired pancreatic beta cell function may contribute to the increased risk of glucose intolerance among adult offspring born to women with diabetes during pregnancy....

Oxidative Metabolism Genes Are Not Responsive to Oxidative Stress in Rodent Beta Cell Lines

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Faer Morrison

2012-01-01

Full Text Available Altered expression of oxidative metabolism genes has been described in the skeletal muscle of individuals with type 2 diabetes. Pancreatic beta cells contain low levels of antioxidant enzymes and are particularly susceptible to oxidative stress. In this study, we explored the effect of hyperglycemia-induced oxidative stress on a panel of oxidative metabolism genes in a rodent beta cell line. We exposed INS-1 rodent beta cells to low (5.6 mmol/L, ambient (11 mmol/L, and high (28 mmol/L glucose conditions for 48 hours. Increases in oxidative stress were measured using the fluorescent probe dihydrorhodamine 123. We then measured the expression levels of a panel of 90 oxidative metabolism genes by real-time PCR. Elevated reactive oxygen species (ROS production was evident in INS-1 cells after 48 hours (P<0.05. TLDA analysis revealed a significant (P<0.05 upregulation of 16 of the 90 genes under hyperglycemic conditions, although these expression differences did not reflect differences in ROS. We conclude that although altered glycemia may influence the expression of some oxidative metabolism genes, this effect is probably not mediated by increased ROS production. The alterations to the expression of oxidative metabolism genes previously observed in human diabetic skeletal muscle do not appear to be mirrored in rodent pancreatic beta cells.

Pancreas-Specific Sirt1-Deficiency in Mice Compromises Beta-Cell Function without Development of Hyperglycemia.

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Pinho, Andreia V; Bensellam, Mohammed; Wauters, Elke; Rees, Maxine; Giry-Laterriere, Marc; Mawson, Amanda; Ly, Le Quan; Biankin, Andrew V; Wu, Jianmin; Laybutt, D Ross; Rooman, Ilse

2015-01-01

Sirtuin 1 (Sirt1) has been reported to be a critical positive regulator of glucose-stimulated insulin secretion in pancreatic beta-cells. The effects on islet cells and blood glucose levels when Sirt1 is deleted specifically in the pancreas are still unclear. This study examined islet glucose responsiveness, blood glucose levels, pancreatic islet histology and gene expression in Pdx1Cre; Sirt1ex4F/F mice that have loss of function and loss of expression of Sirt1 specifically in the pancreas. We found that in the Pdx1Cre; Sirt1ex4F/F mice, the relative insulin positive area and the islet size distribution were unchanged. However, beta-cells were functionally impaired, presenting with lower glucose-stimulated insulin secretion. This defect was not due to a reduced expression of insulin but was associated with a decreased expression of the glucose transporter Slc2a2/Glut2 and of the Glucagon like peptide-1 receptor (Glp1r) as well as a marked down regulation of endoplasmic reticulum (ER) chaperones that participate in the Unfolded Protein Response (UPR) pathway. Counter intuitively, the Sirt1-deficient mice did not develop hyperglycemia. Pancreatic polypeptide (PP) cells were the only other islet cells affected, with reduced numbers in the Sirt1-deficient pancreas. This study provides new mechanistic insights showing that beta-cell function in Sirt1-deficient pancreas is affected due to altered glucose sensing and deregulation of the UPR pathway. Interestingly, we uncovered a context in which impaired beta-cell function is not accompanied by increased glycemia. This points to a unique compensatory mechanism. Given the reduction in PP, investigation of its role in the control of blood glucose is warranted.

Pancreas-Specific Sirt1-Deficiency in Mice Compromises Beta-Cell Function without Development of Hyperglycemia.

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Andreia V Pinho

Full Text Available Sirtuin 1 (Sirt1 has been reported to be a critical positive regulator of glucose-stimulated insulin secretion in pancreatic beta-cells. The effects on islet cells and blood glucose levels when Sirt1 is deleted specifically in the pancreas are still unclear.This study examined islet glucose responsiveness, blood glucose levels, pancreatic islet histology and gene expression in Pdx1Cre; Sirt1ex4F/F mice that have loss of function and loss of expression of Sirt1 specifically in the pancreas.We found that in the Pdx1Cre; Sirt1ex4F/F mice, the relative insulin positive area and the islet size distribution were unchanged. However, beta-cells were functionally impaired, presenting with lower glucose-stimulated insulin secretion. This defect was not due to a reduced expression of insulin but was associated with a decreased expression of the glucose transporter Slc2a2/Glut2 and of the Glucagon like peptide-1 receptor (Glp1r as well as a marked down regulation of endoplasmic reticulum (ER chaperones that participate in the Unfolded Protein Response (UPR pathway. Counter intuitively, the Sirt1-deficient mice did not develop hyperglycemia. Pancreatic polypeptide (PP cells were the only other islet cells affected, with reduced numbers in the Sirt1-deficient pancreas.This study provides new mechanistic insights showing that beta-cell function in Sirt1-deficient pancreas is affected due to altered glucose sensing and deregulation of the UPR pathway. Interestingly, we uncovered a context in which impaired beta-cell function is not accompanied by increased glycemia. This points to a unique compensatory mechanism. Given the reduction in PP, investigation of its role in the control of blood glucose is warranted.

Efficient Generation of Glucose-Responsive Beta Cells from Isolated GP2+ Human Pancreatic Progenitors

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Jacqueline Ameri

2017-04-01

Full Text Available Stem cell-based therapy for type 1 diabetes would benefit from implementation of a cell purification step at the pancreatic endoderm stage. This would increase the safety of the final cell product, allow the establishment of an intermediate-stage stem cell bank, and provide a means for upscaling β cell manufacturing. Comparative gene expression analysis revealed glycoprotein 2 (GP2 as a specific cell surface marker for isolating pancreatic endoderm cells (PECs from differentiated hESCs and human fetal pancreas. Isolated GP2+ PECs efficiently differentiated into glucose responsive insulin-producing cells in vitro. We found that in vitro PEC proliferation declines due to enhanced expression of the cyclin-dependent kinase (CDK inhibitors CDKN1A and CDKN2A. However, we identified a time window when reducing CDKN1A or CDKN2A expression increased proliferation and yield of GP2+ PECs. Altogether, our results contribute tools and concepts toward the isolation and use of PECs as a source for the safe production of hPSC-derived β cells.

Pancreatic beta-cell function is a stronger predictor of changes in glycemic control after an aerobic exercise intervention than insulin sensitivity

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Solomon, Thomas; Malin, Steven K; Karstoft, Kristian

2013-01-01

glucose-stimulated insulin secretion (GSIS), and disposition index (DI) were measured following 12-16-weeks of aerobic exercise training. Regression analyses were used to identify relationships between variables.ResultsFollowing training, 86% of subjects increased VO2max and lost weight. HbA1c, fasting......ContextUnderstanding inter-subject variability in glycemic control following exercise training will help individualize treatment.ObjectiveTo determine whether this variability is related to training-induced changes in insulin sensitivity or pancreatic beta-cell function.Design, Setting....... Training increased first- and second-phase DI in 83% and 74% of subjects. Training-induced changes in glycemic control were related to changes in GSIS (P...

Lactate overrides central nervous but not beta-cell glucose sensing in humans.

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Schmid, Sebastian M; Jauch-Chara, Kamila; Hallschmid, Manfred; Oltmanns, Kerstin M; Peters, Achim; Born, Jan; Schultes, Bernd

2008-12-01

Lactate has been shown to serve as an alternative energy substrate in the central nervous system and to interact with hypothalamic glucose sensors. On the background of marked similarities between central nervous and beta-cell glucose sensing, we examined whether lactate also interacts with pancreatic glucose-sensing mechanisms in vivo. The effects of intravenously infused lactate vs placebo (saline) on central nervous and pancreatic glucose sensing were assessed during euglycemic and hypoglycemic clamp experiments in 10 healthy men. The release of neuroendocrine counterregulatory hormones during hypoglycemia was considered to reflect central nervous glucose sensing, whereas endogenous insulin secretion as assessed by serum C-peptide levels served as an indicator of pancreatic beta-cell glucose sensing. Lactate infusion blunted the counterregulatory hormonal responses to hypoglycemia, in particular, the release of epinephrine (P = .007) and growth hormone (P = .004), so that higher glucose infusion rates (P = .012) were required to maintain the target blood glucose levels. In contrast, the decrease in C-peptide concentrations during the hypoglycemic clamp remained completely unaffected by lactate (P = .60). During euglycemic clamp conditions, lactate infusion did not affect the concentrations of C-peptide and of counterregulatory hormones, with the exception of norepinephrine levels that were lower during lactate than saline infusion (P = .049) independently of the glycemic condition. Data indicate that glucose sensing of beta-cells is specific to glucose, whereas glucose sensing at the central nervous level can be overridden by lactate, reflecting the brain's ability to rely on lactate as an alternative major energy source.

Interactions between the metabolism of L-leucine and D-glucose in the pancreatic. beta. -cells

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Gylfe, E; Sehlin, J [Umeaa Univ. (Sweden). Dept. of Histology

1976-01-01

..beta..-cell-rich pancreatic islets microdissected from obese-hyperglycemic mice were used to study interactions between the metabolism of L-leucine and D-glucose. L-leucine reduced the islet content of aspartic acid whereas D-glucose, when added to L-leucine-incubated islets, increased the contents of aspartic acid and ..gamma..-aminobutyric acid (GABA). D-glucose also increased the incorporation of L-leucine carbon into aspartic acid, GABA and glutamic acid, suggesting stimulation of a malate shuttle mechanism. When expressed per mole of the individual amino acids, the incorporation of L-leucine carbon into GABA was 2.5 - 4 times higher than into glutamic acid indicating intracellular compartmentation of the latter amino acid. Both L-leucine and D-leucine stimulated /sup 14/CO/sub 2/ production from /sup 14/C-labelled D-glucose. L-leucine did not affect /sup 3/H/sub 2/O production from tritiated D-glucose. The present data do not indicate a role of other amino acids or D-glucose in L-leucine-stimulated insulin release.

T Cell-Mediated Beta Cell Destruction: Autoimmunity and Alloimmunity in the Context of Type 1 Diabetes

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Adam L. Burrack

2017-12-01

Full Text Available Type 1 diabetes (T1D results from destruction of pancreatic beta cells by T cells of the immune system. Despite improvements in insulin analogs and continuous blood glucose level monitoring, there is no cure for T1D, and some individuals develop life-threatening complications. Pancreas and islet transplantation have been attractive therapeutic approaches; however, transplants containing insulin-producing cells are vulnerable to both recurrent autoimmunity and conventional allograft rejection. Current immune suppression treatments subdue the immune system, but not without complications. Ideally a successful approach would target only the destructive immune cells and leave the remaining immune system intact to fight foreign pathogens. This review discusses the autoimmune diabetes disease process, diabetic complications that warrant a transplant, and alloimmunity. First, we describe the current understanding of autoimmune destruction of beta cells including the roles of CD4 and CD8 T cells and several possibilities for antigen-specific tolerance induction. Second, we outline diabetic complications necessitating beta cell replacement. Third, we discuss transplant recognition, potential sources for beta cell replacement, and tolerance-promoting therapies under development. We hypothesize that a better understanding of autoreactive T cell targets during disease pathogenesis and alloimmunity following transplant destruction could enhance attempts to re-establish tolerance to beta cells.

Role of 3-Hydroxy Fatty Acid-Induced Hepatic Lipotoxicity in Acute Fatty Liver of Pregnancy

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Sathish Kumar Natarajan

2018-01-01

Full Text Available Acute fatty liver of pregnancy (AFLP, a catastrophic illness for both the mother and the unborn offspring, develops in the last trimester of pregnancy with significant maternal and perinatal mortality. AFLP is also recognized as an obstetric and medical emergency. Maternal AFLP is highly associated with a fetal homozygous mutation (1528G>C in the gene that encodes for mitochondrial long-chain hydroxy acyl-CoA dehydrogenase (LCHAD. The mutation in LCHAD results in the accumulation of 3-hydroxy fatty acids, such as 3-hydroxy myristic acid, 3-hydroxy palmitic acid and 3-hydroxy dicarboxylic acid in the placenta, which are then shunted to the maternal circulation leading to the development of acute liver injury observed in patients with AFLP. In this review, we will discuss the mechanistic role of increased 3-hydroxy fatty acid in causing lipotoxicity to the liver and in inducing oxidative stress, mitochondrial dysfunction and hepatocyte lipoapoptosis. Further, we also review the role of 3-hydroxy fatty acids in causing placental damage, pancreatic islet β-cell glucolipotoxicity, brain damage, and retinal epithelial cells lipoapoptosis in patients with LCHAD deficiency.

Role of 3-Hydroxy Fatty Acid-Induced Hepatic Lipotoxicity in Acute Fatty Liver of Pregnancy

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Ibdah, Jamal A.

2018-01-01

Acute fatty liver of pregnancy (AFLP), a catastrophic illness for both the mother and the unborn offspring, develops in the last trimester of pregnancy with significant maternal and perinatal mortality. AFLP is also recognized as an obstetric and medical emergency. Maternal AFLP is highly associated with a fetal homozygous mutation (1528G>C) in the gene that encodes for mitochondrial long-chain hydroxy acyl-CoA dehydrogenase (LCHAD). The mutation in LCHAD results in the accumulation of 3-hydroxy fatty acids, such as 3-hydroxy myristic acid, 3-hydroxy palmitic acid and 3-hydroxy dicarboxylic acid in the placenta, which are then shunted to the maternal circulation leading to the development of acute liver injury observed in patients with AFLP. In this review, we will discuss the mechanistic role of increased 3-hydroxy fatty acid in causing lipotoxicity to the liver and in inducing oxidative stress, mitochondrial dysfunction and hepatocyte lipoapoptosis. Further, we also review the role of 3-hydroxy fatty acids in causing placental damage, pancreatic islet β-cell glucolipotoxicity, brain damage, and retinal epithelial cells lipoapoptosis in patients with LCHAD deficiency. PMID:29361796

Plasticity of adult human pancreatic duct cells by neurogenin3-mediated reprogramming.

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Nathalie Swales

Full Text Available AIMS/HYPOTHESIS: Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3. In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it. METHODS: The extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or addition of pancreatic endocrine transcription factors Myt1, MafA and Pdx1 we intended to improve the reprogramming. RESULTS: Ngn3 stimulates duct cells to express a focused set of genes that are characteristic for islet endocrine cells and/or neural tissues. This neuro-endocrine shift however, is incomplete with less than 10% of full duct-to-endocrine reprogramming achieved. Transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene. Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1. CONCLUSIONS/INTERPRETATION: The results provide further insight into the plasticity of adult human duct cells and suggest measurable routes to enhance Ngn3-mediated in vitro reprogramming protocols for regenerative beta cell therapy in diabetes.

Pancreatic Fat Is Associated With Metabolic Syndrome and Visceral Fat but Not Beta-Cell Function or Body Mass Index in Pediatric Obesity.

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Staaf, Johan; Labmayr, Viktor; Paulmichl, Katharina; Manell, Hannes; Cen, Jing; Ciba, Iris; Dahlbom, Marie; Roomp, Kirsten; Anderwald, Christian-Heinz; Meissnitzer, Matthias; Schneider, Reinhard; Forslund, Anders; Widhalm, Kurt; Bergquist, Jonas; Ahlström, Håkan; Bergsten, Peter; Weghuber, Daniel; Kullberg, Joel

2017-03-01

Adolescents with obesity have increased risk of type 2 diabetes and metabolic syndrome (MetS). Pancreatic fat has been related to these conditions; however, little is known about associations in pediatric obesity. The present study was designed to explore these associations further. We examined 116 subjects, 90 with obesity. Anthropometry, MetS, blood samples, and oral glucose tolerance tests were assessed using standard techniques. Pancreatic fat fraction (PFF) and other fat depots were quantified using magnetic resonance imaging. The PFF was elevated in subjects with obesity. No association between PFF and body mass index-standard deviation score (BMI-SDS) was found in the obesity subcohort. Pancreatic fat fraction correlated to Insulin Secretion Sensitivity Index-2 and Homeostatic Model Assessment of Insulin Resistance in simple regression; however, when using adjusted regression and correcting for BMI-SDS and other fat compartments, PFF correlated only to visceral adipose tissue and fasting glucose. Highest levels of PFF were found in subjects with obesity and MetS. In adolescents with obesity, PFF is elevated and associated to MetS, fasting glucose, and visceral adipose tissue but not to beta-cell function, glucose tolerance, or BMI-SDS. This study demonstrates that conclusions regarding PFF and its associations depend on the body mass features of the cohort.

Efficient Generation of NKX6-1+ Pancreatic Progenitors from Multiple Human Pluripotent Stem Cell Lines

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M. Cristina Nostro

2015-04-01

Full Text Available Human pluripotent stem cells (hPSCs represent a renewable source of pancreatic beta cells for both basic research and therapeutic applications. Given this outstanding potential, significant efforts have been made to identify the signaling pathways that regulate pancreatic development in hPSC differentiation cultures. In this study, we demonstrate that the combination of epidermal growth factor (EGF and nicotinamide signaling induces the generation of NKX6-1+ progenitors from all hPSC lines tested. Furthermore, we show that the size of the NKX6-1+ population is regulated by the duration of treatment with retinoic acid, fibroblast growth factor 10 (FGF10, and inhibitors of bone morphogenetic protein (BMP and hedgehog signaling pathways. When transplanted into NOD scid gamma (NSG recipients, these progenitors differentiate to give rise to exocrine and endocrine cells, including monohormonal insulin+ cells. Together, these findings provide an efficient and reproducible strategy for generating highly enriched populations of hPSC-derived beta cell progenitors for studies aimed at further characterizing their developmental potential in vivo and deciphering the pathways that regulate their maturation in vitro.

cPLA2alpha-evoked formation of arachidonic acid and lysophospholipids is required for exocytosis in mouse pancreatic beta-cells

DEFF Research Database (Denmark)

Juhl, Kirstine; Høy, Marianne; Olsen, Hervør L

2003-01-01

Using capacitance measurements, we investigated the effects of intracellularly applied recombinant human cytosolic phospholipase A2 (cPLA2alpha) and its lipolytic products arachidonic acid and lysophosphatidylcholine on Ca2+-dependent exocytosis in single mouse pancreatic beta-cells. cPLA2alpha...... from 70-80 to 280-300. cPLA2alpha-stimulated exocytosis was antagonized by the specific cPLA2 inhibitor AACOCF3. Ca2+-evoked exocytosis was reduced by 40% in cells treated with AACOCF3 or an antisense oligonucleotide against cPLA2alpha. The action of cPLA2alpha was mimicked by a combination...... of arachidonic acid and lysophosphatidylcholine (470% stimulation) in which each compound alone doubled the exocytotic response. Priming of insulin-containing secretory granules has been reported to involve Cl- uptake through ClC-3 Cl- channels. Accordingly, the stimulatory action of cPLA2alpha was inhibited...

Lack of TXNIP protects against mitochondria-mediated apoptosis but not against fatty acid-induced ER stress-mediated beta-cell death.

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Chen, Junqin; Fontes, Ghislaine; Saxena, Geetu; Poitout, Vincent; Shalev, Anath

2010-02-01

We have previously shown that lack of thioredoxin-interacting protein (TXNIP) protects against diabetes and glucotoxicity-induced beta-cell apoptosis. Because the role of TXNIP in lipotoxicity is unknown, the goal of the present study was to determine whether TXNIP expression is regulated by fatty acids and whether TXNIP deficiency also protects beta-cells against lipoapoptosis. RESARCH DESIGN AND METHODS: To determine the effects of fatty acids on beta-cell TXNIP expression, INS-1 cells and isolated islets were incubated with/without palmitate and rats underwent cyclic infusions of glucose and/or Intralipid prior to islet isolation and analysis by quantitative real-time RT-PCR and immunoblotting. Using primary wild-type and TXNIP-deficient islets, we then assessed the effects of palmitate on apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL]), mitochondrial death pathway (cytochrome c release), and endoplasmic reticulum (ER) stress (binding protein [BiP], C/EBP homologous protein [CHOP]). Effects of TXNIP deficiency were also tested in the context of staurosporine (mitochondrial damage) or thapsigargin (ER stress). Glucose elicited a dramatic increase in islet TXNIP expression both in vitro and in vivo, whereas fatty acids had no such effect and, when combined with glucose, even abolished the glucose effect. We also found that TXNIP deficiency does not effectively protect against palmitate or thapsigargin-induced beta-cell apoptosis, but specifically prevents staurosporine- or glucose-induced toxicity. Our results demonstrate that unlike glucose, fatty acids do not induce beta-cell expression of proapoptotic TXNIP. They further reveal that TXNIP deficiency specifically inhibits the mitochondrial death pathway underlying beta-cell glucotoxicity, whereas it has very few protective effects against ER stress-mediated lipoapoptosis.

Human beta-cell precursors mature into functional insulin-producing cells in an immunoisolation device: implications for diabetes cell therapies.

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Lee, Seung-Hee; Hao, Ergeng; Savinov, Alexei Y; Geron, Ifat; Strongin, Alex Y; Itkin-Ansari, Pamela

2009-04-15

Islet transplantation is limited by the need for chronic immunosuppression and the paucity of donor tissue. As new sources of human beta-cells are developed (e.g., stem cell-derived tissue), transplanting them in a durable device could obviate the need for immunosuppression, while also protecting the patient from any risk of tumorigenicity. Here, we studied (1) the survival and function of encapsulated human beta-cells and their progenitors and (2) the engraftment of encapsulated murine beta-cells in allo- and autoimmune settings. Human islets and human fetal pancreatic islet-like cell clusters were encapsulated in polytetrafluorethylene devices (TheraCyte) and transplanted into immunodeficient mice. Graft survival and function was measured by immunohistochemistry, circulating human C-peptide levels, and blood glucose levels. Bioluminescent imaging was used to monitor encapsulated neonatal murine islets. Encapsulated human islet-like cell clusters survived, replicated, and acquired a level of glucose responsive insulin secretion sufficient to ameliorate hyperglycemia in diabetic mice. Bioluminescent imaging of encapsulated murine neonatal islets revealed a dynamic process of cell death followed by regrowth, resulting in robust long-term allograft survival. Further, in the non-obese diabetic (NOD) mouse model of type I diabetes, encapsulated primary beta-cells ameliorated diabetes without stimulating a detectable T-cell response. We demonstrate for the first time that human beta-cells function is compatible with encapsulation in a durable, immunoprotective device. Moreover, our study suggests that encapsulation of beta-cells before terminal differentiation will be a successful approach for new cell-based therapies for diabetes, such as those derived from stem cells.

Dendritic Cells Promote Pancreatic Viability in Mice with Acute Pancreatitis

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Bedrosian, Andrea S.; Nguyen, Andrew H.; Hackman, Michael; Connolly, Michael K.; Malhotra, Ashim; Ibrahim, Junaid; Cieza-Rubio, Napoleon E.; Henning, Justin R.; Barilla, Rocky; Rehman, Adeel; Pachter, H. Leon; Medina-Zea, Marco V.; Cohen, Steven M.; Frey, Alan B.; Acehan, Devrim; Miller, George

2011-01-01

Background & Aims Acute pancreatitis increases morbidity and mortality from organ necrosis by mechanisms that are incompletely understood. Dendritic cells (DCs) can promote or suppress inflammation, depending on their subtype and context. We investigated the roles of DC in development of acute pancreatitis. Methods Acute pancreatitis was induced in CD11c.DTR mice using caerulein or L-arginine; DCs were depleted by administration of diphtheria toxin. Survival was analyzed using Kaplan-Meier analysis. Results Numbers of MHC II+CD11c+DC increased 100-fold in pancreas of mice with acute pancreatitis, to account for nearly 15% of intra-pancreatic leukocytes. Intra-pancreatic DC acquired an immune phenotype in mice with acute pancreatitis; they expressed higher levels of MHC II and CD86 and increased production of interleukin-6, membrane cofactor protein (MCP)-1, and tumor necrosis factor (TNF)-α. However, rather than inducing an organ-destructive inflammatory process, DC were required for pancreatic viability; the exocrine pancreas died in mice that were depleted of DC and challenged with caerulein or L-arginine. All mice with pancreatitis that were depleted of DC died from acinar cell death within 4 days. Depletion of DC from mice with pancreatitis resulted in neutrophil infiltration and increased levels of systemic markers of inflammation. However, the organ necrosis associated with depletion of DC did not require infiltrating neutrophils, activation of NF-κB, or signaling by mitogen-activated protein kinase or TNF-α. Conclusions DC are required for pancreatic viability in mice with acute pancreatitis and might protect organs against cell stress. PMID:21801698

Obesity as a clinical and public health problem: is there a need for a new definition based on lipotoxicity effects?

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Sørensen, Thorkild I A; Virtue, Sam; Vidal-Puig, Antonio

2010-01-01

mortality. The lipotoxicity theory implies that the fat stored in droplets of triglycerides in the cells are biologically inert and that the metabolic dysfunctions are primarily due to the increased exposure of the cells to fatty acids. If this is true, it has profound implications for the interpretations...... to avoid excess exposure of the cells to the fatty acids before the metabolic dysfunctions have emerged. In analogy with the glucose tolerance test, a fatty acid tolerance test may be needed to identify individuals who are at a level of risk for developing lipotoxicity induced metabolic dysfunctions...

Bone morphogenetic protein 4 inhibits insulin secretion from rodent beta cells through regulation of calbindin1 expression and reduced voltage-dependent calcium currents

DEFF Research Database (Denmark)

Christensen, Gitte L.; Jacobsen, Maria L. B.; Wendt, Anna

2015-01-01

AIMS/HYPOTHESIS: Type 2 diabetes is characterised by progressive loss of pancreatic beta cell mass and function. Therefore, it is of therapeutic interest to identify factors with the potential to improve beta cell proliferation and insulin secretion. Bone morphogenetic protein 4 (BMP4) expression...

Pancreatic effect of andrographolide isolated from Andrographis paniculata (Burm. f.) Nees.

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Nugroho, Agung Endro; Rais, Ichwan Ridwan; Setiawan, Iwan; Pratiwi, Pramita Yuli; Hadibarata, Tony; Tegar, Maulana; Pramono, Suwidjiyo

2014-01-01

Andrographis paniculata (Burm. f.) Nees is a plant that originates from India and grows widely to Southeast which used for several purposes mainly as treatment of diabetes mellitus so the aim of this study was evaluate andrographolide for its pancreatic effect in neonatal streptozotocin (STZ)-induced diabetic rats, a model of type 2 diabetic rats. Diabetic condition was induced with an intraperitoneal injection of 90 mg kg(-1) streptozotocin in two-day-old rats. After three months, the neonatal STZ-induced diabetic rats were treated with andrographolide or andrographolide-enriched extract of A. paniculata (AEEAP) for 8 consecutive days. Pancreatic effect was evaluated by estimating mainly the preprandial and postprandial blood glucose levels and other parameters such as morphology of pancreatic islet, beta cells density and morphology and immunohistochemically pancreatic insulin. Andrographolide significantly (p < 0.05) decreased the levels of blood glucose and improved diabetic rat islet and beta cells. However, AEEAP exhibited moderate hypoglycaemic effects on the blood glucose levels. Moderate changes in beta cells were observed after AEEAP treatment. They could restore decreasing of pancreatic insulin contents. Based on these results andrographolide and AEEAP exhibited pancreatic actions in neonatal STZ-induced diabetic rats. The activity of andrographolide was more effective than this of AEEAP.

Zinc as a paracrine effector in pancreatic islet cell death.

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Kim, B J; Kim, Y H; Kim, S; Kim, J W; Koh, J Y; Oh, S H; Lee, M K; Kim, K W; Lee, M S

2000-03-01

Because of a huge amount of Zn2+ in secretory granules of pancreatic islet beta-cells, Zn2+ released in certain conditions might affect the function or survival of islet cells. We studied potential paracrine effects of endogenous Zn2+ on beta-cell death. Zn2+ induced insulinoma/islet cell death in a dose-dependent manner. Chelation of released endogenous Zn2+ by CaEDTA significantly decreased streptozotocin (STZ)-induced islet cell death in an in vitro culture system simulating in vivo circumstances but not in the conventional culture system. Zn2+ chelation in vivo by continuous CaEDTA infusion significantly decreased the incidence of diabetes after STZ administration. N-(6-methoxy-quinolyl)-para-toluene-sulfonamide staining revealed that Zn2+ was densely deposited in degenerating islet cells 24 h after STZ treatment, which was decreased by CaEDTA infusion. We show here that Zn2+ is not a passive element for insulin storage but an active participant in islet cell death in certain conditions, which in time might contribute to the development of diabetes in aged people.

Effect of long-term transfusion therapy on the glycometabolic status and pancreatic beta cell function in patients with beta Thalassemia major

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Kamalakshi G Bhat

2014-01-01

Full Text Available Background: Diabetes mellitus is a major complication of iron overload in patients with beta thalassemia major. Design: This is a descriptive study conducted in a Tertiary Care Teaching Hospital to analyze beta cell function and insulin resistance, and their relation to iron overload status in beta thalassemia major. Fasting glucose, two-hour post load glucose, fasting insulin, alanine amino transaminase (ALT, and ferritin were used as outcome measures. The homeostatic model assessment (HOMA model was used to calculate the beta cell function and insulin resistance index. Results: Of the 30 cases, 20% had impaired fasting glucose, 3.3% had impaired glucose tolerance, and none had diabetes. Fasting glucose was not significant between the cases and controls (P = 0.113. Fasting insulin (P = 0.001, ferritin (P = 0.001, and ALT (P = 0.001 levels were significantly high in the cases. Insulin resistance index was significantly higher in the cases (P = 0.001 as also the beta cell function (P = 0.001. With increase in age and the number of units transfused there is a decline in beta cell function, fasting insulin, and insulin resistance after attaining the maximum level. This suggests that initial insulin resistance is followed by insulin depletion due to loss of beta cell function, leading to diabetes mellitus. Conclusion: Impaired glucose tolerance (IGT and insulin resistance precede the onset of insulin-dependent diabetes and adequate chelation therapy is essential for delaying the onset or for prevention of diabetes.

Rising intracellular zinc by membrane depolarization and glucose in insulin-secreting clonal HIT-T15 beta cells.

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Slepchenko, Kira G; Li, Yang V

2012-01-01

Zinc (Zn(2+)) appears to be intimately involved in insulin metabolism since insulin secretion is correlated with zinc secretion in response to glucose stimulation, but little is known about the regulation of zinc homeostasis in pancreatic beta-cells. This study set out to identify the intracellular zinc transient by imaging free cytosolic zinc in HIT-T15 beta-cells with fluorescent zinc indicators. We observed that membrane depolarization by KCl (30-60 mM) was able to induce a rapid increase in cytosolic concentration of zinc. Multiple zinc transients of similar magnitude were elicited during repeated stimulations. The amplitude of zinc responses was not affected by the removal of extracellular calcium or zinc. However, the half-time of the rising slope was significantly slower after removing extracellular zinc with zinc chelator CaEDTA, suggesting that extracellular zinc affect the initial rising phase of zinc response. Glucose (10 mM) induced substantial and progressive increases in intracellular zinc concentration in a similar way as KCl, with variation in the onset and the duration of zinc mobilization. It is known that the depolarization of beta-cell membrane is coupled with the secretion of insulin. Rising intracellular zinc concentration may act as a critical signaling factor in insulin metabolism of pancreatic beta-cells.

Role of pancreatic polypeptide as a market of transplanted insulin-producing fetal pig cells.

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Tuch, B E; Tabiin, M T; Casamento, F M; Yao, M; Georges, P; Amaratunga, A; Pinto, A N

2001-01-01

Transplantation of insulin-producing fetal pancreatic tissue into diabetic recipients has been shown to normalize blood glucose levels after several months. This time period is required for the growth and maturation of the fetal tissue so insulin levels cannot be used as a marker of graft function while the beta-cell is immature. Therefore, we have examined the use of another pancreatic endocrine hormone, pancreatic polypeptide (PP), to monitor graft function. The cell that produces this hormone has been shown to be the first mature endocrine cell in the fetal pancreas. Fetal pig pancreatic tissue, both in the form of 1 mm3 explants and islet-like cell clusters (ICCs), was transplanted into immunodeficient SCID mice and the levels of PP and insulin were measured in plasma and in the graft for up to 12 weeks. PP was detected in the untransplanted explants (0.58 pmol/mg) and ICCs (0.06 pmol/ICC) and the PP to insulin ratio was 2.7% and 5.8%, respectively. PP (but not porcine C-peptide, a marker of insulin secretion) was detectable in the plasma of SCID mice from 4 days to 3 weeks after transplantation, but not thereafter. The highest values were obtained at 4 days to 1 week. In the grafted tissue PP and insulin were present at all time points and the ratio of PP to insulin was 59%, 87%, 75%, 56%, 7%, 8%, and 7% at 4 days, 1, 2, 3, 6, 9, and 12 weeks, respectively. The decline in PP levels 3 weeks after transplantation was associated with beta-cell development in the graft. PP was also secreted by fetal pig pancreatic explants transplanted into diabetic NOD/SCID mice, with plasma levels measurable in the first week after the tissue was grafted. In immunocompetent BALB/c mice transplanted with the tissue, PP was detectable in plasma for 2 days after transplantation but not at 4 days, when cellular rejection commenced, or thereafter. We conclude that plasma PP levels can be used as a marker of the viability of fetal porcine pancreatic tissue in the first 3 weeks after

Small molecule kaempferol modulates PDX-1 protein expression and subsequently promotes pancreatic β-cell survival and function via CREB

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Zhang, Yanling.; Zhen, Wei.; Maechler, Pierre; Liu, Dongmin

2013-01-01

Chronic hyperlipidemia causes β-cell apoptosis and dysfunction, thereby contributing to the pathogenesis of T2D. Thus, searching for agents to promote pancreatic β-cell survival and improve its function could be a promising strategy to prevent and treat T2D. We investigated the effects of kaempferol, a small molecule isolated from ginkgo biloba, on apoptosis and function of β-cells and further determined the mechanism underlying its actions. Kaempferol treatment promoted viability, inhibited apoptosis, and reduced caspase-3 activity in INS-1E cells and human islets chronically exposed to palmitate. In addition, kaempferol prevented the lipotoxicity-induced down-regulation of anti-apoptotic proteins Akt and Bcl-2. The cytoprotective effects of kaempferol were associated with improved insulin secretion, synthesis, and PDX-1 expression. Chronic hyperlipidemia significantly diminished cAMP production, PKA activation, and CREB phosphorylation and its regulated transcriptional activity in β-cells, all of which were restored by kaempferol treatment. Disruption of CREB expression by transfection of CREB siRNA in INS-1E cells or adenoviral transfer of dominant-negative forms of CREB in human islets ablated kaempferol protection of β-cell apoptosis and dysfunction caused by palmitate. Incubation of INS-1E cells or human islets with kaempferol for 48 h induced PDX-1 expression. This effect of kaempferol on PDX-1 expression was not shared by a host of structurally related flavonoid compounds. PDX-1 gene knockdown reduced kaempferol–stimulated cAMP generation and CREB activation in INS-1E cells. These findings demonstrate that kaempferol is a novel survivor factor for pancreatic β-cells via up-regulating the PDX-1/cAMP/PKA/CREB signaling cascade. PMID:22819546

An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells

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Massumi, Mohammad; Pourasgari, Farzaneh; Nalla, Amarnadh

2016-01-01

developed an abbreviated five-stage protocol (25-30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems......The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have...... positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux...

Deletion of glutamate dehydrogenase in beta-cells abolishes part of the insulin secretory response not required for glucose homeostasis

DEFF Research Database (Denmark)

Carobbio, Stefania; Frigerio, Francesca; Rubi, Blanca

2009-01-01

Insulin exocytosis is regulated in pancreatic ss-cells by a cascade of intracellular signals translating glucose levels into corresponding secretory responses. The mitochondrial enzyme glutamate dehydrogenase (GDH) is regarded as a major player in this process, although its abrogation has not been...... tested yet in animal models. Here, we generated transgenic mice, named betaGlud1(-/-), with ss-cell-specific GDH deletion. Our results show that GDH plays an essential role in the full development of the insulin secretory response. In situ pancreatic perfusion revealed that glucose-stimulated insulin...... secretion was reduced by 37% in betaGlud1(-/-). Furthermore, isolated islets with either constitutive or acute adenovirus-mediated knock-out of GDH showed a 49 and 38% reduction in glucose-induced insulin release, respectively. Adenovirus-mediated re-expression of GDH in betaGlud1(-/-) islets fully restored...

Inflammatory stress of pancreatic beta cells drives release of extracellular heat-shock protein 90α.

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Ocaña, Gail J; Pérez, Liliana; Guindon, Lynette; Deffit, Sarah N; Evans-Molina, Carmella; Thurmond, Debbie C; Blum, Janice S

2017-06-01

A major obstacle in predicting and preventing the development of autoimmune type 1 diabetes (T1D) in at-risk individuals is the lack of well-established early biomarkers indicative of ongoing beta cell stress during the pre-clinical phase of disease. Recently, serum levels of the α cytoplasmic isoform of heat-shock protein 90 (hsp90) were shown to be elevated in individuals with new-onset T1D. We therefore hypothesized that hsp90α could be released from beta cells in response to cellular stress and inflammation associated with the earliest stages of T1D. Here, human beta cell lines and cadaveric islets released hsp90α in response to stress induced by treatment with a combination of pro-inflammatory cytokines including interleukin-1β, tumour necrosis factor-α and interferon-γ. Mechanistically, hsp90α release was found to be driven by cytokine-induced endoplasmic reticulum stress mediated by c-Jun N-terminal kinase (JNK), a pathway that can eventually lead to beta cell apoptosis. Cytokine-induced beta cell hsp90α release and JNK activation were significantly reduced by pre-treating cells with the endoplasmic reticulum stress-mitigating chemical chaperone tauroursodeoxycholic acid. The hsp90α release by cells may therefore be a sensitive indicator of stress during inflammation and a useful tool in assessing therapeutic mitigation of cytokine-induced cell damage linked to autoimmunity. © 2017 John Wiley & Sons Ltd.

The role of polyunsaturated fatty acids (n-3 PUFAs) on the pancreatic β-cells and insulin action.

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Baynes, Habtamu Wondifraw; Mideksa, Seifu; Ambachew, Sintayehu

2018-03-14

Polyunsaturated Fatty acids have multiple effects in peripheral tissues and pancreatic beta cell function. The n-3 Polyunsaturated Fatty acids prevent and reverse high-fat-diet induced adipose tissue inflammation and insulin resistance. Insulin secretion is stimulated by glucose, amino acids, and glucagon- like peptide-1 in tissue containing high levels of n-3 Polyunsaturated Fatty acids than lower level of n-3 Polyunsaturated Fatty acids. Also, n-3 Polyunsaturated Fatty acids led to decreased production of prostaglandin, which in turn contributed to the elevation of insulin secretion. N-3 polyunsaturated fatty acids prevent cytokine-induced cell death in pancreatic islets. Supplementation of n-3 Polyunsaturated Fatty acids for human subjects prevent beta cell destruction and insulin resistance. It also enhances insulin secretion, reduction in lipid profiles and glucose concentration particularly in type II diabetes patients. Therefore there should be a focus on the treatment mechanism of insulin related obesity and diabetes by n-3 polyunsaturated fatty acids.

B cell depletion reduces T cell activation in pancreatic islets in a murine autoimmune diabetes model.

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Da Rosa, Larissa C; Boldison, Joanne; De Leenheer, Evy; Davies, Joanne; Wen, Li; Wong, F Susan

2018-06-01

Type 1 diabetes is a T cell-mediated autoimmune disease characterised by the destruction of beta cells in the islets of Langerhans, resulting in deficient insulin production. B cell depletion therapy has proved successful in preventing diabetes and restoring euglycaemia in animal models of diabetes, as well as in preserving beta cell function in clinical trials in the short term. We aimed to report a full characterisation of B cell kinetics post B cell depletion, with a focus on pancreatic islets. Transgenic NOD mice with a human CD20 transgene expressed on B cells were injected with an anti-CD20 depleting antibody. B cells were analysed using multivariable flow cytometry. There was a 10 week delay in the onset of diabetes when comparing control and experimental groups, although the final difference in the diabetes incidence, following prolonged observation, was not statistically significant (p = 0.07). The co-stimulatory molecules CD80 and CD86 were reduced on stimulation of B cells during B cell depletion and repopulation. IL-10-producing regulatory B cells were not induced in repopulated B cells in the periphery, post anti-CD20 depletion. However, the early depletion of B cells had a marked effect on T cells in the local islet infiltrate. We demonstrated a lack of T cell activation, specifically with reduced CD44 expression and effector function, including IFN-γ production from both CD4 + and CD8 + T cells. These CD8 + T cells remained altered in the pancreatic islets long after B cell depletion and repopulation. Our findings suggest that B cell depletion can have an impact on T cell regulation, inducing a durable effect that is present long after repopulation. We suggest that this local effect of reducing autoimmune T cell activity contributes to delay in the onset of autoimmune diabetes.

Clinical characteristics and beta cell function in Chinese patients with newly diagnosed type 2 diabetes mellitus with different levels of serum triglyceride.

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Zheng, Shuang; Zhou, Huan; Han, Tingting; Li, Yangxue; Zhang, Yao; Liu, Wei; Hu, Yaomin

2015-04-29

To explore clinical characteristics and beta cell function in Chinese patients with newly diagnosed drug naive type 2 diabetes mellitus (T2DM) with different levels of serum triglyceride (TG). Patients with newly diagnosed T2DM (n = 624) were enrolled and divided into different groups according to levels of serum TG. All patients underwent oral glucose tolerance tests and insulin releasing tests. Demographic data, lipid profiles, glucose levels, and insulin profiles were compared between different groups. Basic insulin secretion function index (homeostasis model assessment for beta cell function index, HOMA-β), modified beta cell function index (MBCI), glucose disposition indices (DI), and early insulin secretion function index (insulinogenic index, IGI) were used to evaluate the beta cell function. Patients of newly diagnosed T2DM with hypertriglyceridemia were younger, fatter and had worse lipid profiles, glucose profiles, and high insulin levels than those with normal TG. There is no difference in early phase insulin secretion among groups of newly diagnosed T2DM patients with different TG levels. The basal beta cell function (HOMA-β and MBCI) initially increased along rising TG levels and then decreased as the TG levels rose further. The insulin sensitivity was relatively high in patients with a low level of TG and low with a high level of TG. Hypertriglyceridemia influences clinical characteristics and β cell function of Chinese patients with newly diagnosed T2DM. A better management of dyslipidemia may, to some extent, reduce the effect of lipotoxicity, thereby improving glucose homeostasis in patients with newly diagnosed T2DM.

Rac1-NADPH oxidase signaling promotes CD36 activation under glucotoxic conditions in pancreatic beta cells.

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Elumalai, Suma; Karunakaran, Udayakumar; Lee, In Kyu; Moon, Jun Sung; Won, Kyu Chang

2017-04-01

We recently reported that cluster determinant 36 (CD36), a fatty acid transporter, plays a pivotal role in glucotoxicity-induced β-cell dysfunction. However, little is known about how glucotoxicity influences CD36 expression. Emerging evidence suggests that the small GTPase Rac1 is involved in the pathogenesis of beta cell dysfunction in type 2 diabetes (T2D). The primary objective of the current study was to determine the role of Rac1 in CD36 activation and its impact on β-cell dysfunction in diabetes mellitus. To address this question, we subjected INS-1 cells and human beta cells (1.1B4) to high glucose conditions (30mM) in the presence or absence of Rac1 inhibition either by NSC23766 (Rac1 GTPase inhibitor) or small interfering RNA. High glucose exposure in INS-1 and human beta cells (1.1b4) resulted in the activation of Rac1 and induced cell apoptosis. Rac1 activation mediates NADPH oxidase (NOX) activation leading to elevated ROS production in both cells. Activation of the Rac1-NOX complex by high glucose levels enhanced CD36 expression in INS-1 and human 1.1b4 beta cell membrane fractions. The inhibition of Rac1 by NSC23766 inhibited NADPH oxidase activity and ROS generation induced by high glucose concentrations in INS-1 & human 1.1b4 beta cells. Inhibition of Rac1-NOX complex activation by NSC23766 significantly reduced CD36 expression in INS-1 and human 1.1b4 beta cell membrane fractions. In addition, Rac1 inhibition by NSC23766 significantly reduced high glucose-induced mitochondrial dysfunction. Furthermore, NADPH oxidase inhibition by VAS2870 also attenuated high glucose-induced ROS generation and cell apoptosis. These results suggest that Rac1-NADPH oxidase dependent CD36 expression contributes to high glucose-induced beta cell dysfunction and cell death. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

A human beta cell line with drug inducible excision of immortalizing transgenes

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Benazra, Marion; Lecomte, Marie-José; Colace, Claire; Müller, Andreas; Machado, Cécile; Pechberty, Severine; Bricout-Neveu, Emilie; Grenier-Godard, Maud; Solimena, Michele; Scharfmann, Raphaël; Czernichow, Paul; Ravassard, Philippe

2015-01-01

Objectives Access to immortalized human pancreatic beta cell lines that are phenotypically close to genuine adult beta cells, represent a major tool to better understand human beta cell physiology and develop new therapeutics for Diabetes. Here we derived a new conditionally immortalized human beta cell line, EndoC-βH3 in which immortalizing transgene can be efficiently removed by simple addition of tamoxifen. Methods We used lentiviral mediated gene transfer to stably integrate a tamoxifen inducible form of CRE (CRE-ERT2) into the recently developed conditionally immortalized EndoC βH2 line. The resulting EndoC-βH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion. Results We showed that EndoC-βH3 expressing CRE-ERT2 can be massively amplified in culture. We established an optimized tamoxifen treatment to efficiently excise the immortalizing transgenes resulting in proliferation arrest. In addition, insulin expression raised by 12 fold and insulin content increased by 23 fold reaching 2 μg of insulin per million cells. Such massive increase was accompanied by enhanced insulin secretion upon glucose stimulation. We further observed that tamoxifen treated cells maintained a stable function for 5 weeks in culture. Conclusions EndoC βH3 cell line represents a powerful tool that allows, using a simple and efficient procedure, the massive production of functional non-proliferative human beta cells. Such cells are close to genuine human beta cells and maintain a stable phenotype for 5 weeks in culture. PMID:26909308

LGR5 and Nanog identify stem cell signature of pancreas beta cells which initiate pancreatic cancer.

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Amsterdam, Abraham; Raanan, Calanit; Schreiber, Letizia; Polin, Nava; Givol, David

2013-04-05

Pancreas cancer, is the fourth leading cause of cancer death but its cell of origin is controversial. We compared the localization of stem cells in normal and cancerous pancreas using antibodies to the stem cell markers Nanog and LGR5. Here we show, for the first time, that LGR5 is expressed in normal pancreas, exclusively in the islets of Langerhans and it is co-localized, surprisingly, with Nanog and insulin in clusters of beta cells. In cancerous pancreas Nanog and LGR5 are expressed in the remaining islets and in all ductal cancer cells. We observed insulin staining among the ductal cancer cells, but not in metastases. This indicates that the islet's beta cells, expressing LGR5 and Nanog markers are the initiating cells of pancreas cancer, which migrated from the islets to form the ductal cancerous tissue, probably after mutation and de-differentiation. This discovery may facilitate treatment of this devastating cancer. Copyright © 2013 Elsevier Inc. All rights reserved.

Intermittent fasting preserves beta-cell mass in obesity-induced diabetes via the autophagy-lysosome pathway.

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Liu, Haiyan; Javaheri, Ali; Godar, Rebecca J; Murphy, John; Ma, Xiucui; Rohatgi, Nidhi; Mahadevan, Jana; Hyrc, Krzysztof; Saftig, Paul; Marshall, Connie; McDaniel, Michael L; Remedi, Maria S; Razani, Babak; Urano, Fumihiko; Diwan, Abhinav

2017-01-01

Obesity-induced diabetes is characterized by hyperglycemia, insulin resistance, and progressive beta cell failure. In islets of mice with obesity-induced diabetes, we observe increased beta cell death and impaired autophagic flux. We hypothesized that intermittent fasting, a clinically sustainable therapeutic strategy, stimulates autophagic flux to ameliorate obesity-induced diabetes. Our data show that despite continued high-fat intake, intermittent fasting restores autophagic flux in islets and improves glucose tolerance by enhancing glucose-stimulated insulin secretion, beta cell survival, and nuclear expression of NEUROG3, a marker of pancreatic regeneration. In contrast, intermittent fasting does not rescue beta-cell death or induce NEUROG3 expression in obese mice with lysosomal dysfunction secondary to deficiency of the lysosomal membrane protein, LAMP2 or haplo-insufficiency of BECN1/Beclin 1, a protein critical for autophagosome formation. Moreover, intermittent fasting is sufficient to provoke beta cell death in nonobese lamp2 null mice, attesting to a critical role for lysosome function in beta cell homeostasis under fasting conditions. Beta cells in intermittently-fasted LAMP2- or BECN1-deficient mice exhibit markers of autophagic failure with accumulation of damaged mitochondria and upregulation of oxidative stress. Thus, intermittent fasting preserves organelle quality via the autophagy-lysosome pathway to enhance beta cell survival and stimulates markers of regeneration in obesity-induced diabetes.

Developmental programming of polycystic ovary syndrome (PCOS): prenatal androgens establish pancreatic islet α/β cell ratio and subsequent insulin secretion.

Science.gov (United States)

Ramaswamy, S; Grace, C; Mattei, A A; Siemienowicz, K; Brownlee, W; MacCallum, J; McNeilly, A S; Duncan, W C; Rae, M T

2016-06-06

Exogenous androgenic steroids applied to pregnant sheep programmes a PCOS-like phenotype in female offspring. Via ultrasound guidance we applied steroids directly to ovine fetuses at d62 and d82 of gestation, and examined fetal (day 90 gestation) and postnatal (11 months old) pancreatic structure and function. Of three classes of steroid agonists applied (androgen - Testosterone propionate (TP), estrogen - Diethystilbesterol (DES) and glucocorticoid - Dexamethasone (DEX)), only androgens (TP) caused altered pancreatic development. Beta cell numbers were significantly elevated in prenatally androgenised female fetuses (P = 0.03) (to approximately the higher numbers found in male fetuses), whereas alpha cell counts were unaffected, precipitating decreased alpha:beta cell ratios in the developing fetal pancreas (P = 0.001), sustained into adolescence (P = 0.0004). In adolescence basal insulin secretion was significantly higher in female offspring from androgen-excess pregnancies (P = 0.045), and an exaggerated, hyperinsulinaemic response to glucose challenge (P = 0.0007) observed, whereas prenatal DES or DEX treatment had no effects upon insulin secretion. Postnatal insulin secretion correlated with beta cell numbers (P = 0.03). We conclude that the pancreas is a primary locus of androgenic stimulation during development, giving rise to postnatal offspring whose pancreas secreted excess insulin due to excess beta cells in the presence of a normal number of alpha cells.

Lysine deacetylases are produced in pancreatic beta cells and are differentially regulated by proinflammatory cytokines

DEFF Research Database (Denmark)

Lundh, M; Christensen, D P; Rasmussen, D N

2010-01-01

Cytokine-induced beta cell toxicity is abrogated by non-selective inhibitors of lysine deacetylases (KDACs). The KDAC family consists of 11 members, namely histone deacetylases HDAC1 to HDAC11, but it is not known which KDAC members play a role in cytokine-mediated beta cell death. The aim...

Simultaneous characterization of pancreatic stellate cells and other pancreatic components within three-dimensional tissue environment during chronic pancreatitis

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Hu, Wenyan; Fu, Ling

2013-05-01

Pancreatic stellate cells (PSCs) and other pancreatic components that play a critical role in exocrine pancreatic diseases are generally identified separately by conventional studies, which provide indirect links between these components. Here, nonlinear optical microscopy was evaluated for simultaneous characterization of these components within a three-dimensional (3-D) tissue environment, primarily based on multichannel detection of intrinsic optical emissions and cell morphology. Fresh rat pancreatic tissues harvested at 1 day, 7 days, and 28 days after induction of chronic pancreatitis were imaged, respectively. PSCs, inflammatory cells, blood vessels, and collagen fibers were identified simultaneously. The PSCs at day 1 of chronic pancreatitis showed significant enlargement compared with those in normal pancreas (ppancreatic components coincidently within 3-D pancreatic tissues. It is a prospect for intravital observation of dynamic events under natural physiological conditions, and might help uncover the key mechanisms of exocrine pancreatic diseases, leading to more effective treatments.

Pancreatic Islet Cell Transplantation

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Warnock, Garth L.; Rajotte, Ray V.

1992-01-01

Transplantation of insulin-producing tissue offers a physiologic approach to restoration of glycemic control. Whereas transplantation of vascularized pancreatic grafts has recently achieved encouraging results, pancreatic islet cell transplantation holds the promise of low morbidity and reduced requirements for agressive immunosuppression for recipients. Islet cell transplantation was recently demonstrated to induce euglycemia with insulin independence. Imagesp1656-a PMID:21221366

Rising Intracellular Zinc by Membrane Depolarization and Glucose in Insulin-Secreting Clonal HIT-T15 Beta Cells

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Kira G. Slepchenko

2012-01-01

Full Text Available Zinc (Zn2+ appears to be intimately involved in insulin metabolism since insulin secretion is correlated with zinc secretion in response to glucose stimulation, but little is known about the regulation of zinc homeostasis in pancreatic beta-cells. This study set out to identify the intracellular zinc transient by imaging free cytosolic zinc in HIT-T15 beta-cells with fluorescent zinc indicators. We observed that membrane depolarization by KCl (30–60 mM was able to induce a rapid increase in cytosolic concentration of zinc. Multiple zinc transients of similar magnitude were elicited during repeated stimulations. The amplitude of zinc responses was not affected by the removal of extracellular calcium or zinc. However, the half-time of the rising slope was significantly slower after removing extracellular zinc with zinc chelator CaEDTA, suggesting that extracellular zinc affect the initial rising phase of zinc response. Glucose (10 mM induced substantial and progressive increases in intracellular zinc concentration in a similar way as KCl, with variation in the onset and the duration of zinc mobilization. It is known that the depolarization of beta-cell membrane is coupled with the secretion of insulin. Rising intracellular zinc concentration may act as a critical signaling factor in insulin metabolism of pancreatic beta-cells.

Palmitate activates autophagy in INS-1E β-cells and in isolated rat and human pancreatic islets.

Directory of Open Access Journals (Sweden)

Luisa Martino

Full Text Available We have investigated the in vitro effects of increased levels of glucose and free fatty acids on autophagy activation in pancreatic beta cells. INS-1E cells and isolated rat and human pancreatic islets were incubated for various times (from 2 to 24 h at different concentrations of glucose and/or palmitic acid. Then, cell survival was evaluated and autophagy activation was explored by using various biochemical and morphological techniques. In INS-1E cells as well as in rat and human islets, 0.5 and 1.0 mM palmitate markedly increased autophagic vacuole formation, whereas high glucose was ineffective alone and caused little additional change when combined with palmitate. Furthermore, LC3-II immunofluorescence co-localized with that of cathepsin D, a lysosomal marker, showing that the autophagic flux was not hampered in PA-treated cells. These effects were maintained up to 18-24 h incubation and were associated with a significant decline of cell survival correlated with both palmitate concentration and incubation time. Ultrastructural analysis showed that autophagy activation, as evidenced by the occurrence of many autophagic vacuoles in the cytoplasm of beta cells, was associated with a diffuse and remarkable swelling of the endoplasmic reticulum. Our results indicate that among the metabolic alterations typically associated with type 2 diabetes, high free fatty acids levels could play a role in the activation of autophagy in beta cells, through a mechanism that might involve the induction of endoplasmic reticulum stress.

Stabilization of beta-catenin induces pancreas tumor formation.

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Heiser, Patrick W; Cano, David A; Landsman, Limor; Kim, Grace E; Kench, James G; Klimstra, David S; Taketo, Maketo M; Biankin, Andrew V; Hebrok, Matthias

2008-10-01

beta-Catenin signaling within the canonical Wnt pathway is essential for pancreas development. However, the pathway is normally down-regulated in the adult organ. Increased cytoplasmic and nuclear localization of beta-catenin can be detected in nearly all human solid pseudopapillary neoplasms (SPN), a rare tumor with low malignant potential. Conversely, pancreatic ductal adenocarcinoma (PDA) accounts for the majority of pancreatic tumors and is among the leading causes of cancer death. Whereas activating mutations within beta-catenin and other members of the canonical Wnt pathway are rare, recent reports have implicated Wnt signaling in the development and progression of human PDA. Here, we sought to address the role of beta-catenin signaling in pancreas tumorigenesis. Using Cre/lox technology, we conditionally activated beta-catenin in a subset of murine pancreatic cells in vivo. Activation of beta-catenin results in the formation of large pancreatic tumors at a high frequency in adult mice. These tumors resemble human SPN based on morphologic and immunohistochemical comparisons. Interestingly, stabilization of beta-catenin blocks the formation of pancreatic intraepithelial neoplasia (PanIN) in the presence of an activating mutation in Kras that is known to predispose individuals to PDA. Instead, mice in which beta-catenin and Kras are concurrently activated develop distinct ductal neoplasms that do not resemble PanIN lesions. These results demonstrate that activation of beta-catenin is sufficient to induce pancreas tumorigenesis. Moreover, they indicate that the sequence in which oncogenic mutations are acquired has profound consequences on the phenotype of the resulting tumor.

Adipose tissue-derived stem cells promote pancreatic cancer cell proliferation and invasion

International Nuclear Information System (INIS)

Ji, S.Q.; Cao, J.; Zhang, Q.Y.; Li, Y.Y.; Yan, Y.Q.; Yu, F.X.

2013-01-01

To explore the effects of adipose tissue-derived stem cells (ADSCs) on the proliferation and invasion of pancreatic cancer cells in vitro and the possible mechanism involved, ADSCs were cocultured with pancreatic cancer cells, and a cell counting kit (CCK-8) was used to detect the proliferation of pancreatic cancer cells. ELISA was used to determine the concentration of stromal cell-derived factor-1 (SDF-1) in the supernatants. RT-PCR was performed to detect the expression of the chemokine receptor CXCR4 in pancreatic cancer cells and ADSCs. An in vitro invasion assay was used to measure invasion of pancreatic cancer cells. SDF-1 was detected in the supernatants of ADSCs, but not in pancreatic cancer cells. Higher CXCR4 mRNA levels were detected in the pancreatic cancer cell lines compared with ADSCs (109.3±10.7 and 97.6±7.6 vs 18.3±1.7, respectively; P<0.01). In addition, conditioned medium from ADSCs promoted the proliferation and invasion of pancreatic cancer cells, and AMD3100, a CXCR4 antagonist, significantly downregulated these growth-promoting effects. We conclude that ADSCs can promote the proliferation and invasion of pancreatic cancer cells, which may involve the SDF-1/CXCR4 axis

Adipose tissue-derived stem cells promote pancreatic cancer cell proliferation and invasion

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Ji, S.Q.; Cao, J. [Department of Liver Surgery I, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai (China); Zhang, Q.Y.; Li, Y.Y. [Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Wenzhou Medical College, Wenzhou (China); Yan, Y.Q. [Department of Liver Surgery I, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai (China); Yu, F.X. [Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Wenzhou Medical College, Wenzhou (China)

2013-09-27

To explore the effects of adipose tissue-derived stem cells (ADSCs) on the proliferation and invasion of pancreatic cancer cells in vitro and the possible mechanism involved, ADSCs were cocultured with pancreatic cancer cells, and a cell counting kit (CCK-8) was used to detect the proliferation of pancreatic cancer cells. ELISA was used to determine the concentration of stromal cell-derived factor-1 (SDF-1) in the supernatants. RT-PCR was performed to detect the expression of the chemokine receptor CXCR4 in pancreatic cancer cells and ADSCs. An in vitro invasion assay was used to measure invasion of pancreatic cancer cells. SDF-1 was detected in the supernatants of ADSCs, but not in pancreatic cancer cells. Higher CXCR4 mRNA levels were detected in the pancreatic cancer cell lines compared with ADSCs (109.3±10.7 and 97.6±7.6 vs 18.3±1.7, respectively; P<0.01). In addition, conditioned medium from ADSCs promoted the proliferation and invasion of pancreatic cancer cells, and AMD3100, a CXCR4 antagonist, significantly downregulated these growth-promoting effects. We conclude that ADSCs can promote the proliferation and invasion of pancreatic cancer cells, which may involve the SDF-1/CXCR4 axis.

Pancreatic Cancer-Derived Exosomes Cause Paraneoplastic β-cell Dysfunction.

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Javeed, Naureen; Sagar, Gunisha; Dutta, Shamit K; Smyrk, Thomas C; Lau, Julie S; Bhattacharya, Santanu; Truty, Mark; Petersen, Gloria M; Kaufman, Randal J; Chari, Suresh T; Mukhopadhyay, Debabrata

2015-04-01

Pancreatic cancer frequently causes diabetes. We recently proposed adrenomedullin as a candidate mediator of pancreatic β-cell dysfunction in pancreatic cancer. How pancreatic cancer-derived adrenomedullin reaches β cells remote from the cancer to induce β-cell dysfunction is unknown. We tested a novel hypothesis that pancreatic cancer sheds adrenomedullin-containing exosomes into circulation, which are transported to β cells and impair insulin secretion. We characterized exosomes from conditioned media of pancreatic cancer cell lines (n = 5) and portal/peripheral venous blood of patients with pancreatic cancer (n = 20). Western blot analysis showed the presence of adrenomedullin in pancreatic cancer-exosomes. We determined the effect of adrenomedullin-containing pancreatic cancer exosomes on insulin secretion from INS-1 β cells and human islets, and demonstrated the mechanism of exosome internalization into β cells. We studied the interaction between β-cell adrenomedullin receptors and adrenomedullin present in pancreatic cancer-exosomes. In addition, the effect of adrenomedullin on endoplasmic reticulum (ER) stress response genes and reactive oxygen/nitrogen species generation in β cells was shown. Exosomes were found to be the predominant extracellular vesicles secreted by pancreatic cancer into culture media and patient plasma. Pancreatic cancer-exosomes contained adrenomedullin and CA19-9, readily entered β cells through caveolin-mediated endocytosis or macropinocytosis, and inhibited insulin secretion. Adrenomedullin in pancreatic cancer exosomes interacted with its receptor on β cells. Adrenomedullin receptor blockade abrogated the inhibitory effect of exosomes on insulin secretion. β cells exposed to adrenomedullin or pancreatic cancer exosomes showed upregulation of ER stress genes and increased reactive oxygen/nitrogen species. Pancreatic cancer causes paraneoplastic β-cell dysfunction by shedding adrenomedullin(+)/CA19-9(+) exosomes into

Impact of fetal and neonatal environment on beta cell function and development of diabetes

DEFF Research Database (Denmark)

Nielsen, Jens H; Haase, Tobias N; Jaksch, Caroline

2014-01-01

on the beta cells in both the mother and the fetus and how various conditions like diabetes, obesity, overnutrition and undernutrition during and after pregnancy may influence the ability of the offspring to adapt to changes in insulin demand later in life. The influence of environmental factors including...... that the intrauterine environment during pregnancy has an impact on the gene expression that may persist until adulthood and cause metabolic diseases like obesity and type 2 diabetes. As the pancreatic beta cells are crucial in the regulation of metabolism this article will describe the influence of normal pregnancy...... nutrients and gut microbiota on appetite regulation, mitochondrial activity and the immune system that may affect beta cell growth and function directly and indirectly is discussed. The possible role of epigenetic changes in the transgenerational transmission of the adverse programming may be the most...

Curcumin Modulates Pancreatic Adenocarcinoma Cell-Derived Exosomal Function

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Osterman, Carlos J. Diaz; Lynch, James C.; Leaf, Patrick; Gonda, Amber; Ferguson Bennit, Heather R.; Griffiths, Duncan; Wall, Nathan R.

2015-01-01

Pancreatic cancer has the highest mortality rates of all cancer types. One potential explanation for the aggressiveness of this disease is that cancer cells have been found to communicate with one another using membrane-bound vesicles known as exosomes. These exosomes carry pro-survival molecules and increase the proliferation, survival, and metastatic potential of recipient cells, suggesting that tumor-derived exosomes are powerful drivers of tumor progression. Thus, to successfully address and eradicate pancreatic cancer, it is imperative to develop therapeutic strategies that neutralize cancer cells and exosomes simultaneously. Curcumin, a turmeric root derivative, has been shown to have potent anti-cancer and anti-inflammatory effects in vitro and in vivo. Recent studies have suggested that exosomal curcumin exerts anti-inflammatory properties on recipient cells. However, curcumin’s effects on exosomal pro-tumor function have yet to be determined. We hypothesize that curcumin will alter the pro-survival role of exosomes from pancreatic cancer cells toward a pro-death role, resulting in reduced cell viability of recipient pancreatic cancer cells. The main objective of this study was to determine the functional alterations of exosomes released by pancreatic cancer cells exposed to curcumin compared to exosomes from untreated pancreatic cancer cells. We demonstrate, using an in vitro cell culture model involving pancreatic adenocarcinoma cell lines PANC-1 and MIA PaCa-2, that curcumin is incorporated into exosomes isolated from curcumin-treated pancreatic cancer cells as observed by spectral studies and fluorescence microscopy. Furthermore, curcumin is delivered to recipient pancreatic cancer cells via exosomes, promoting cytotoxicity as demonstrated by Hoffman modulation contrast microscopy as well as AlamarBlue and Trypan blue exclusion assays. Collectively, these data suggest that the efficacy of curcumin may be enhanced in pancreatic cancer cells through

Lipid droplets induced by secreted phospholipase A2 and unsaturated fatty acids protect breast cancer cells from nutrient and lipotoxic stress.

Science.gov (United States)

Jarc, Eva; Kump, Ana; Malavašič, Petra; Eichmann, Thomas O; Zimmermann, Robert; Petan, Toni

2018-03-01

Cancer cells driven by the Ras oncogene scavenge unsaturated fatty acids (FAs) from their environment to counter nutrient stress. The human group X secreted phospholipase A 2 (hGX sPLA 2 ) releases FAs from membrane phospholipids, stimulates lipid droplet (LD) biogenesis in Ras-driven triple-negative breast cancer (TNBC) cells and enables their survival during starvation. Here we examined the role of LDs, induced by hGX sPLA 2 and unsaturated FAs, in protection of TNBC cells against nutrient stress. We found that hGX sPLA 2 releases a mixture of unsaturated FAs, including ω-3 and ω-6 polyunsaturated FAs (PUFAs), from TNBC cells. Starvation-induced breakdown of LDs induced by low micromolar concentrations of unsaturated FAs, including PUFAs, was associated with protection from cell death. Interestingly, adipose triglyceride lipase (ATGL) contributed to LD breakdown during starvation, but it was not required for the pro-survival effects of hGX sPLA 2 and unsaturated FAs. High micromolar concentrations of PUFAs, but not OA, induced oxidative stress-dependent cell death in TNBC cells. Inhibition of triacylglycerol (TAG) synthesis suppressed LD biogenesis and potentiated PUFA-induced cell damage. On the contrary, stimulation of LD biogenesis by hGX sPLA 2 and suppression of LD breakdown by ATGL depletion reduced PUFA-induced oxidative stress and cell death. Finally, lipidomic analyses revealed that sequestration of PUFAs in LDs by sPLA 2 -induced TAG remodelling and retention of PUFAs in LDs by inhibition of ATGL-mediated TAG lipolysis protect from PUFA lipotoxicity. LDs are thus antioxidant and pro-survival organelles that guard TNBC cells against nutrient and lipotoxic stress and emerge as attractive targets for novel therapeutic interventions. Copyright © 2017 Elsevier B.V. All rights reserved.

Impaired fatty acid oxidation as a cause for lipotoxicity in cardiomyocytes

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Haffar, T. [Université de Montreal (Canada); Montreal Heart Institute (Canada); Bérubé-Simard, F. [Montreal Heart Institute (Canada); Bousette, N., E-mail: nicolas.bousette@umontreal.ca [Université de Montreal (Canada); Montreal Heart Institute (Canada)

2015-12-04

A major cause for diabetic cardiomyopathy is excess lipid accumulation. To elucidate mechanisms of lipotoxicity mediated diabetic heart disease we need to further our understanding of how lipid metabolism is altered in the diabetic heart. Here we investigated the role of lipid clearance by oxidation as a regulator of lipid-mediated toxicity (lipotoxicity). We evaluated the effect of pre-treating rat neonatal cardiomyocytes (NCMs) with either oleate (mono-unsaturated fatty acid) or palmitate (saturated fatty acid) on fatty acid oxidation (FAO) by measuring {sup 14}C–CO{sub 2} production. We evaluated carnitine palmitoyltransferase (Cpt1b) expression by western blotting and mitochondrial membrane potential by quantitative and qualitative fluorescence analyses using the JC-1 dye. We inhibited the Cpt1b pharmacologically using etomoxir and genetically by knocking down its expression using LentiVector mediated transduction of siRNAs targeting the Cpt1b gene. We found that palmitate had a slower clearance rate from NCMs than oleate, and this was associated with a significant decrease in FAO. This impairment in FAO was not the result of either loss of Cpt1b protein or mitochondrial integrity. Enhancing FAO with either oleate or carnitine was associated with a significant attenuation of palmitate mediated lipotoxicity. In contrast impairing FAO in oleate treated NCMs caused lipotoxicity. Here we demonstrate that a major difference between non-toxic unsaturated fatty acids and toxic saturated fatty acids is there ability to stimulate or inhibit fatty acid oxidation, respectively. This has important implications for diabetic cardiomyopathy since diabetic hearts consistently exhibit elevated lipid accumulation. - Highlights: • Palmitate had a slower clearance rate from NCMs than oleate. • Palmitate caused a significant decrease in fatty acid oxidation in cardiomyocytes. • Impaired FAO was not due to loss of Cpt1b protein or mitochondrial integrity. • Enhancing FAO

Impaired fatty acid oxidation as a cause for lipotoxicity in cardiomyocytes

International Nuclear Information System (INIS)

Haffar, T.; Bérubé-Simard, F.; Bousette, N.

2015-01-01

A major cause for diabetic cardiomyopathy is excess lipid accumulation. To elucidate mechanisms of lipotoxicity mediated diabetic heart disease we need to further our understanding of how lipid metabolism is altered in the diabetic heart. Here we investigated the role of lipid clearance by oxidation as a regulator of lipid-mediated toxicity (lipotoxicity). We evaluated the effect of pre-treating rat neonatal cardiomyocytes (NCMs) with either oleate (mono-unsaturated fatty acid) or palmitate (saturated fatty acid) on fatty acid oxidation (FAO) by measuring "1"4C–CO_2 production. We evaluated carnitine palmitoyltransferase (Cpt1b) expression by western blotting and mitochondrial membrane potential by quantitative and qualitative fluorescence analyses using the JC-1 dye. We inhibited the Cpt1b pharmacologically using etomoxir and genetically by knocking down its expression using LentiVector mediated transduction of siRNAs targeting the Cpt1b gene. We found that palmitate had a slower clearance rate from NCMs than oleate, and this was associated with a significant decrease in FAO. This impairment in FAO was not the result of either loss of Cpt1b protein or mitochondrial integrity. Enhancing FAO with either oleate or carnitine was associated with a significant attenuation of palmitate mediated lipotoxicity. In contrast impairing FAO in oleate treated NCMs caused lipotoxicity. Here we demonstrate that a major difference between non-toxic unsaturated fatty acids and toxic saturated fatty acids is there ability to stimulate or inhibit fatty acid oxidation, respectively. This has important implications for diabetic cardiomyopathy since diabetic hearts consistently exhibit elevated lipid accumulation. - Highlights: • Palmitate had a slower clearance rate from NCMs than oleate. • Palmitate caused a significant decrease in fatty acid oxidation in cardiomyocytes. • Impaired FAO was not due to loss of Cpt1b protein or mitochondrial integrity. • Enhancing FAO attenuated

Urtica Dioica Distillate Regenerates Pancreatic Beta Cells in Streptozotocin-Induced Diabetic Rats

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Gohari, Ali; Noorafshan, Ali; Akmali, Masoumeh; Zamani-Garmsiri, Fahimeh; Seghatoleslam, Atefeh

2018-01-01

Background Urtica dioica is known as an anti-hyperglycemic plant. Urtica dioica distillate (UD) is a traditional Iranian drink, locally known as “aragh gazaneh”. In spite of its widespread consumption in Iran, according to traditional Iranian medicine, there is no scientific report on the usefulness of UD for diabetic patients. This survey was designed to evaluate its protective effects for the recovery from diabetes by determining the serum insulin, blood glucose, volume of pancreatic islets, and the number and volume of β-cells in diabetic rats. Methods A total of 48 Sprague-Dawley male rats (200-250 g) were randomly distributed into 6 groups (n=8), including non-diabetic plus distilled water (DW), non-diabetic plus UD, diabetic plus DW, diabetic plus UD, diabetic plus insulin, and diabetic plus glibenclamide. DW, UD, and glibenclamide were administered via intragastric gavage and insulin was injected subcutaneously. After four weeks of experiments, blood samples were collected for serum insulin and blood glucose assay. Pancreas was also evaluated using stereological method. The SPSS software was used for statistical analysis. Kruskal-Wallis, repeated measurements, and Mann-Whitney U test were applied for comparisons between the groups. Results The treatment of diabetic rats with UD reduced the blood glucose dramatically (P<0.001) and increased serum insulin levels significantly (P=0.03) in comparison to the diabetic plus DW rats. Treatment with UD did not affect the mean β-cell volumes in the diabetic rats when compared to the diabetic plus DW rats, but the islet volumes and β-cell numbers were significantly recovered. Conclusion UD treatment in diabetic rats improves hyperglycemia by partially restoring plasma insulin levels. The data suggest that UD prevents islet atrophy and/or regenerate pancreatic β-cells. PMID:29749986

RAS/ERK modulates TGFbeta-regulated PTEN expression in human pancreatic adenocarcinoma cells.

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Chow, Jimmy Y C; Quach, Khai T; Cabrera, Betty L; Cabral, Jennifer A; Beck, Stayce E; Carethers, John M

2007-11-01

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is rarely mutated in pancreatic cancers, but its regulation by transforming growth factor (TGF)-beta might mediate growth suppression and other oncogenic actions. Here, we examined the role of TGFbeta and the effects of oncogenic K-RAS/ERK upon PTEN expression in the absence of SMAD4. We utilized two SMAD4-null pancreatic cell lines, CAPAN-1 (K-RAS mutant) and BxPc-3 (WT-K-RAS), both of which express TGFbeta surface receptors. Cells were treated with TGFbeta1 and separated into cytosolic/nuclear fractions for western blotting with phospho-SMAD2, SMAD 2, 4 phospho-ATP-dependent tyrosine kinases (Akt), Akt and PTEN antibodies. PTEN mRNA levels were assessed by reverse transcriptase-polymerase chain reaction. The MEK1 inhibitor, PD98059, was used to block the downstream action of oncogenic K-RAS/ERK, as was a dominant-negative (DN) K-RAS construct. TGFbeta increased phospho-SMAD2 in both cytosolic and nuclear fractions. PD98059 treatment further increased phospho-SMAD2 in the nucleus of both pancreatic cell lines, and DN-K-RAS further improved SMAD translocation in K-RAS mutant CAPAN cells. TGFbeta treatment significantly suppressed PTEN protein levels concomitant with activation of Akt by 48 h through transcriptional reduction of PTEN mRNA that was evident by 6 h. TGFbeta-induced PTEN suppression was reversed by PD98059 and DN-K-RAS compared with treatments without TGFbeta. TGFbeta-induced PTEN expression was inversely related to cellular proliferation. Thus, oncogenic K-RAS/ERK in pancreatic adenocarcinoma facilitates TGFbeta-induced transcriptional down-regulation of the tumor suppressor PTEN in a SMAD4-independent manner and could constitute a signaling switch mechanism from growth suppression to growth promotion in pancreatic cancers.

Distinct Internalization Pathways of Human Amylin Monomers and Its Cytotoxic Oligomers in Pancreatic Cells

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Trikha, Saurabh; Jeremic, Aleksandar M.

2013-01-01

Toxic human amylin oligomers and aggregates are implicated in the pathogenesis of type 2 diabetes mellitus (TTDM). Although recent studies have shown that pancreatic cells can recycle amylin monomers and toxic oligomers, the exact uptake mechanism and trafficking routes of these molecular forms and their significance for amylin toxicity are yet to be determined. Using pancreatic rat insulinoma (RIN-m5F) beta (β)-cells and human islets as model systems we show that monomers and oligomers cross the plasma membrane (PM) through both endocytotic and non-endocytotic (translocation) mechanisms, the predominance of which is dependent on amylin concentrations and incubation times. At low (≤100 nM) concentrations, internalization of amylin monomers in pancreatic cells is completely blocked by the selective amylin-receptor (AM-R) antagonist, AC-187, indicating an AM-R dependent mechanism. In contrast at cytotoxic (µM) concentrations monomers initially (1 hour) enter pancreatic cells by two distinct mechanisms: translocation and macropinocytosis. However, during the late stage (24 hours) monomers internalize by a clathrin-dependent but AM-R and macropinocytotic independent pathway. Like monomers a small fraction of the oligomers initially enter cells by a non-endocytotic mechanism. In contrast a majority of the oligomers at both early (1 hour) and late times (24 hours) traffic with a fluid-phase marker, dextran, to the same endocytotic compartments, the uptake of which is blocked by potent macropinocytotic inhibitors. This led to a significant increase in extra-cellular PM accumulation, in turn potentiating amylin toxicity in pancreatic cells. Our studies suggest that macropinocytosis is a major but not the only clearance mechanism for both amylin’s molecular forms, thereby serving a cyto-protective role in these cells. PMID:24019897

Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells.

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Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. Copyright © 2015 Elsevier Inc. All rights reserved.

Type 2-diabetes--en heterogen sygdom med kim i fostertilstanden

DEFF Research Database (Denmark)

Vaag, Allan

2012-01-01

Type 2 diabetes (T2D) is a heterogeneous disease with a multifactorial aetiology involving defects in the pancreatic beta cells, liver, muscles, adipose tissue, guts, brain, kidneys and heart. While genetics may only explain a minor proportion of T2D, the contribution of an adverse intrauterine...... environment may take centre stage in the global propagation of T2D. Impaired expandability of subcutaneous adipose tissue in persons with low birthweight may cause T2D due to lipotoxicity in non-adipose organs. Future implications include a stronger focus on individualized treatments in T2D patients...

Elevated androgen levels induce hyperinsulinemia through increase in Ins1 transcription in pancreatic beta cells in female rats.

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Mishra, Jay S; More, Amar S; Kumar, Sathish

2018-01-22

Hyperandrogenism is associated with hyperinsulinemia and insulin resistance in adult females. We tested whether androgens dysregulate pancreatic beta cell function to induce hyperinsulinemia through transcriptional regulation of insulin gene (Ins) in the islets. Adult female Wistar rats implanted with dihydrotestosterone (DHT; 7.5-mg, 90-d release) or placebo pellets were examined after 10 weeks. DHT exposure increased plasma DHT levels by 2-fold similar to that in polycystic ovary syndrome in women. DHT exposure induced hyperinsulinemia with increased HOMA-IR index in fasting state and glucose intolerance and exaggerated insulin responses following glucose tolerance test. DHT females had no change in islet number, size and beta cell proliferation/apoptosis but exhibited significant mitochondrial dysfunction (higher ADP/ATP ratio, decreased mtDNA copy number, increased reactive oxygen production and downregulation of mitochondrial biogenesis) and enhanced glucose-stimulated insulin secretion. Ins expression was increased in DHT islets. In vitro incubation of control islets with DHT dose-dependently stimulated Ins transcription. Analysis of Ins1 gene revealed a putative androgen responsive element in the promoter. Chromatin-immunoprecipitation assays showed that androgen receptors bind to this element in response to DHT stimulation. Furthermore, reporter assays showed that the promoter element is highly responsive to androgens. Insulin stimulated glucose uptake in skeletal muscle was decreased with associated decrease in IRβ expression in DHT females. Our studies identified a novel androgen-mediated mechanism for the control of Ins expression via transcriptional regulation providing a molecular mechanism linking elevated androgens and hyperinsulemia. Decreased IRβ expression in the skeletal muscles may contribute, in part, to glucose intolerance in this model. © The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of

Enterovirus strain and type-specific differences in growth kinetics and virus-induced cell destruction in human pancreatic duct epithelial HPDE cells.

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Smura, Teemu; Natri, Olli; Ylipaasto, Petri; Hellman, Marika; Al-Hello, Haider; Piemonti, Lorenzo; Roivainen, Merja

2015-12-02

Enterovirus infections have been suspected to be involved in the development of type 1 diabetes. However, the pathogenetic mechanism of enterovirus-induced type 1 diabetes is not known. Pancreatic ductal cells are closely associated with pancreatic islets. Therefore, enterovirus infections in ductal cells may also affect beta-cells and be involved in the induction of type 1 diabetes. The aim of this study was to assess the ability of different enterovirus strains to infect, replicate and produce cytopathic effect in human pancreatic ductal cells. Furthermore, the viral factors that affect these capabilities were studied. The pancreatic ductal cells were highly susceptible to enterovirus infections. Both viral growth and cytolysis were detected for several enterovirus serotypes. However, the viral growth and capability to induce cytopathic effect (cpe) did not correlate completely. Some of the virus strains replicated in ductal cells without apparent cpe. Furthermore, there were strain-specific differences in the growth kinetics and the ability to cause cpe within some serotypes. Viral adaptation experiments were carried out to study the potential genetic determinants behind these phenotypic differences. The blind-passage of non-lytic CV-B6-Schmitt strain in HPDE-cells resulted in lytic phenotype and increased progeny production. This was associated with the substitution of a single amino acid (K257E) in the virus capsid protein VP1 and the viral ability to use decay accelerating factor (DAF) as a receptor. This study demonstrates considerable plasticity in the cell tropism, receptor usage and cytolytic properties of enteroviruses and underlines the strong effect of single or few amino acid substitutions in cell tropism and lytic capabilities of a given enterovirus. Since ductal cells are anatomically close to pancreatic islets, the capability of enteroviruses to infect and destroy pancreatic ductal cells may also implicate in respect to enterovirus induced type 1

Sustained beta-cell dysfunction but normalized islet mass in aged thrombospondin-1 deficient mice.

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Carl Johan Drott

Full Text Available Pancreatic islet endothelial cells have in recent years been shown to support beta-cell mass and function by paracrine interactions. Recently, we identified an islets endothelial-specific glycoprotein, thrombospondin-1 (TSP-1, that showed to be of importance for islet angiogenesis and beta-cell function in young mice. The present study aimed to investigate long-term consequences for islet morphology and beta-cell function of TSP-1 deficiency. Islet and beta-cell mass were observed increased at 10-12 weeks of age in TSP-1 deficient mice, but were normalized before 16 weeks of age when compared to wild-type controls. Islet vascularity was normal in 10-12 and 16-week-old TSP-1 deficient animals, whereas islets of one-year-old animals lacking TSP-1 were hypervascular. Beta-cell dysfunction in TSP-1 deficient animals was present at similar magnitudes between 10-12 and 52 weeks of age, as evaluated by glucose tolerance tests. The insulin secretion capacity in vivo of islets in one-year-old TSP-1 deficient animals was only ∼15% of that in wild-type animals. Using a transplantation model, we reconstituted TSP-1 in adult TSP-deficient islets. In contrast to neonatal TSP-1 deficient islets that we previously reported to regain function after TSP-1 reconstitution, adult islets failed to recover. We conclude that TSP-1 deficiency in islets causes changing vascular and endocrine morphological alterations postnatally, but is coupled to a chronic beta-cell dysfunction. The beta-cell dysfunction induced by TSP-1 deficiency is irreversible if not substituted early in life.

Lysosome associated membrane proteins maintain pancreatic acinar cell homeostasis: LAMP-2 deficient mice develop pancreatitis.

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Mareninova, Olga A; Sendler, Matthias; Malla, Sudarshan Ravi; Yakubov, Iskandar; French, Samuel W; Tokhtaeva, Elmira; Vagin, Olga; Oorschot, Viola; Lüllmann-Rauch, Renate; Blanz, Judith; Dawson, David; Klumperman, Judith; Lerch, Markus M; Mayerle, Julia; Gukovsky, Ilya; Gukovskaya, Anna S

2015-11-01

The pathogenic mechanism of pancreatitis is poorly understood. Recent evidence implicates defective autophagy in pancreatitis responses; however, the pathways mediating impaired autophagy in pancreas remain largely unknown. Here, we investigate the role of lysosome associated membrane proteins (LAMPs) in pancreatitis. We analyzed changes in LAMPs in experimental models and human pancreatitis, and the underlying mechanisms: LAMP de-glycosylation and degradation. LAMP cleavage by cathepsin B (CatB) was analyzed by mass spectrometry. We used mice deficient in LAMP-2 to assess its role in pancreatitis. Pancreatic levels of LAMP-1 and LAMP-2 greatly decrease across various pancreatitis models and in human disease. Pancreatitis does not trigger LAMPs' bulk de-glycosylation, but induces their degradation via CatB-mediated cleavage of LAMP molecule close to the boundary between luminal and transmembrane domains. LAMP-2 null mice spontaneously develop pancreatitis that begins with acinar cell vacuolization due to impaired autophagic flux, and progresses to severe pancreas damage characterized by trypsinogen activation, macrophage-driven inflammation, and acinar cell death. LAMP-2 deficiency causes a decrease in pancreatic digestive enzymes content, stimulates the basal and inhibits CCK-induced amylase secretion by acinar cells. The effects of LAMP-2 knockout and acute cerulein pancreatitis overlap, which corroborates the pathogenic role of LAMP decrease in experimental pancreatitis models. The results indicate a critical role for LAMPs, particularly LAMP-2, in maintaining pancreatic acinar cell homeostasis, and provide evidence that defective lysosomal function, resulting in impaired autophagy, leads to pancreatitis. Mice with LAMP-2 deficiency present a novel genetic model of human pancreatitis caused by lysosomal/autophagic dysfunction.

In vivo and in vitro characterization of [18F]-FE-(+)-DTBZ as a tracer for beta-cell mass

International Nuclear Information System (INIS)

Eriksson, Olof; Jahan, Mahabuba; Johnstroem, Peter; Korsgren, Olle; Sundin, Anders; Halldin, Christer; Johansson, Lars

2010-01-01

Introduction: The positron emission tomography (PET) tracer 9-[ 18 F]fluoroethyl-(+)-dihydrotetrabenazine ([ 18 F]-FE-(+)-DTBZ) is a potential candidate for quantifying beta-cell mass in vivo. The purpose was to investigate in vitro and in vivo utility of this tracer for the assessment of beta-cell mass. Methods: Three pigs were intravenously administered [ 18 F]-FE-(+)-DTBZ and examined by PET/computed tomography. Binding parameters were estimated by kinetic modeling. In vitro k D and B max were determined by saturation binding studies of endocrine and exocrine human tissue homogenates. In vitro pancreatic uptake was determined by tissue autoradiography with pancreases from patients with types 1 (T1DM) and 2 diabetes mellitus (T2DM) and healthy controls. Results: [ 18 F]-FE-(+)-DTBZ had a k D of 3.5±1.0 nM, a B max of 382±108 fmol/mg protein and a specificity of 89±1.8% in islet homogenates. The total exocrine uptake was lower and 65% was nondisplaceable. No uptake difference was observed in pancreatic tissue slices from patients with T1DM, T2DM or healthy controls. The in vivo porcine pancreatic uptake reached a peak of standardized uptake value (SUV) of 2.8 with a low distribution volume ratio in all animals. Moderate to high tracer uptake was identified in the bile system and in bone. Conclusions: [ 18 F]-FE-(+)-DTBZ binds to vesicular monoamine transporter 2 (VMAT2) with high specificity in pure islet tissue in vitro. However, there is high nondisplaceable binding to exocrine tissue. In addition, in vivo tracer metabolism and dehalogenation result in severe underestimation of porcine pancreatic VMAT2 expression and BCM. The results do not support [ 18 F]-FE-(+)-DTBZ as a suitable tracer for in vivo beta-cell imaging.

Chemical strategies for pancreatic β cell differentiation, reprogramming, and regeneration.

Science.gov (United States)

Ma, Xiaojie; Zhu, Saiyong

2017-04-01

Generation of unlimited functional pancreatic β cells is critical for the study of pancreatic biology and treatment of diabetes mellitus. Recent advances have suggested several promising directions, including directed differentiation of pancreatic β cells from pluripotent stem cells, reprogramming of pancreatic β cells from other types of somatic cells, and stimulated proliferation and enhanced functions of existing pancreatic β cells. Small molecules are useful in generating unlimited numbers of functional pancreatic cells in vitro and could be further developed as drugs to stimulate endogenous pancreatic regeneration. Here, we provide an updated summary of recent major achievements in pancreatic β cell differentiation, reprogramming, proliferation, and function. These studies will eventually lead to significant advances in the field of pancreatic biology and regeneration. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Cancer Stem Cells in Pancreatic Cancer

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Bao, Qi; Zhao, Yue; Renner, Andrea; Niess, Hanno; Seeliger, Hendrik; Jauch, Karl-Walter; Bruns, Christiane J., E-mail: christiane.bruns@med.uni-muenchen.de [Department of Surgery, Ludwig Maximilian University of Munich, Klinikum Grosshadern, Marchioninistr. 15, D-81377, Munich (Germany)

2010-08-19

Pancreatic cancer is an aggressive malignant solid tumor well-known by early metastasis, local invasion, resistance to standard chemo- and radiotherapy and poor prognosis. Increasing evidence indicates that pancreatic cancer is initiated and propagated by cancer stem cells (CSCs). Here we review the current research results regarding CSCs in pancreatic cancer and discuss the different markers identifying pancreatic CSCs. This review will focus on metastasis, microRNA regulation and anti-CSC therapy in pancreatic cancer.

Is serum zinc associated with pancreatic beta cell function and insulin sensitivity in pre-diabetic and normal individuals? Findings from the Hunter Community Study.

Directory of Open Access Journals (Sweden)

Khanrin P Vashum

Full Text Available AIM: To determine if there is a difference in serum zinc concentration between normoglycaemic, pre-diabetic and type-2 diabetic groups and if this is associated with pancreatic beta cell function and insulin sensitivity in the former 2 groups. METHOD: Cross sectional study of a random sample of older community-dwelling men and women in Newcastle, New South Wales, Australia. Beta cell function, insulin sensitivity and insulin resistance were calculated for normoglycaemic and prediabetes participants using the Homeostasis Model Assessment (HOMA-2 calculator. RESULT: A total of 452 participants were recruited for this study. Approximately 33% (N = 149 had diabetes, 33% (N = 151 had prediabetes and 34% (N = 152 were normoglycaemic. Homeostasis Model Assessment (HOMA parameters were found to be significantly different between normoglycaemic and prediabetes groups (p<0.001. In adjusted linear regression, higher serum zinc concentration was associated with increased insulin sensitivity (p = 0.01 in the prediabetic group. There was also a significant association between smoking and worse insulin sensitivity. CONCLUSION: Higher serum zinc concentration is associated with increased insulin sensitivity. Longitudinal studies are required to determine if low serum zinc concentration plays a role in progression from pre-diabetes to diabetes.

Ubiquitin fold modifier 1 (UFM1 and its target UFBP1 protect pancreatic beta cells from ER stress-induced apoptosis.

Directory of Open Access Journals (Sweden)

Katleen Lemaire

Full Text Available UFM1 is a member of the ubiquitin like protein family. While the enzymatic cascade of UFM1 conjugation has been elucidated in recent years, the biological function remains largely unknown. In this report we demonstrate that the recently identified C20orf116, which we name UFM1-binding protein 1 containing a PCI domain (UFBP1, and CDK5RAP3 interact with UFM1. Components of the UFM1 conjugation pathway (UFM1, UFBP1, UFL1 and CDK5RAP3 are highly expressed in pancreatic islets of Langerhans and some other secretory tissues. Co-localization of UFM1 with UFBP1 in the endoplasmic reticulum (ER depends on UFBP1. We demonstrate that ER stress, which is common in secretory cells, induces expression of Ufm1, Ufbp1 and Ufl1 in the beta-cell line INS-1E. siRNA-mediated Ufm1 or Ufbp1 knockdown enhances apoptosis upon ER stress. Silencing the E3 enzyme UFL1, results in similar outcomes, suggesting that UFM1-UFBP1 conjugation is required to prevent ER stress-induced apoptosis. Together, our data suggest that UFM1-UFBP1 participate in preventing ER stress-induced apoptosis in protein secretory cells.

Cancer Stem Cells in Pancreatic Cancer

Science.gov (United States)

Bao, Qi; Zhao, Yue; Renner, Andrea; Niess, Hanno; Seeliger, Hendrik; Jauch, Karl-Walter; Bruns, Christiane J.

2010-01-01

Pancreatic cancer is an aggressive malignant solid tumor well-known by early metastasis, local invasion, resistance to standard chemo- and radiotherapy and poor prognosis. Increasing evidence indicates that pancreatic cancer is initiated and propagated by cancer stem cells (CSCs). Here we review the current research results regarding CSCs in pancreatic cancer and discuss the different markers identifying pancreatic CSCs. This review will focus on metastasis, microRNA regulation and anti-CSC therapy in pancreatic cancer. PMID:24281178

Cancer Stem Cells in Pancreatic Cancer

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Karl-Walter Jauch

2010-08-01

Full Text Available Pancreatic cancer is an aggressive malignant solid tumor well-known by early metastasis, local invasion, resistance to standard chemo- and radiotherapy and poor prognosis. Increasing evidence indicates that pancreatic cancer is initiated and propagated by cancer stem cells (CSCs. Here we review the current research results regarding CSCs in pancreatic cancer and discuss the different markers identifying pancreatic CSCs. This review will focus on metastasis, microRNA regulation and anti-CSC therapy in pancreatic cancer.

Engineered T cells for pancreatic cancer treatment

Science.gov (United States)

Katari, Usha L; Keirnan, Jacqueline M; Worth, Anna C; Hodges, Sally E; Leen, Ann M; Fisher, William E; Vera, Juan F

2011-01-01

Objective Conventional chemotherapy and radiotherapy produce marginal survival benefits in pancreatic cancer, underscoring the need for novel therapies. The aim of this study is to develop an adoptive T cell transfer approach to target tumours expressing prostate stem cell antigen (PSCA), a tumour-associated antigen that is frequently expressed by pancreatic cancer cells. Methods Expression of PSCA on cell lines and primary tumour samples was confirmed by immunohistochemistry. Healthy donor- and patient-derived T cells were isolated, activated in vitro using CD3/CD28, and transduced with a retroviral vector encoding a chimeric antigen receptor (CAR) targeting PSCA. The ability of these cells to kill tumour cells was analysed by chromium-51 (Cr51) release. Results Prostate stem cell antigen was expressed on >70% of the primary tumour samples screened. Activated, CAR-modified T cells could be readily generated in clinically relevant numbers and were specifically able to kill PSCA-expressing pancreatic cancer cell lines with no non-specific killing of PSCA-negative target cells, thus indicating the potential efficacy and safety of this approach. Conclusions Prostate stem cell antigen is frequently expressed on pancreatic cancer cells and can be targeted for immune-mediated destruction using CAR-modified, adoptively transferred T cells. The safety and efficacy of this approach indicate that it deserves further study and may represent a promising novel treatment for patients with pancreatic cancer. PMID:21843265

CRFR1 is expressed on pancreatic beta cells, promotes beta cell proliferation, and potentiates insulin secretion in a glucose-dependent manner

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Huising, Mark O; van der Meulen, Talitha; Vaughan, Joan M

2009-01-01

Corticotropin-releasing factor (CRF), originally characterized as the principal neuroregulator of the hypothalamus-pituitary-adrenal axis, has broad central and peripheral distribution and actions. We demonstrate the presence of CRF receptor type 1 (CRFR1) on primary beta cells and show that acti...

Enhanced Glucose Tolerance and Pancreatic Beta Cell Function by Low Dose Aspirin in Hyperglycemic Insulin-Resistant Type 2 Diabetic Goto-Kakizaki (GK Rats

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Layla Amiri

2015-07-01

Full Text Available Background/Aim: Type 2 diabetes is the most common metabolic disorder, characterized by insulin resistance and pancreatic islet beta-cell failure. The most common complications associated with type 2 diabetes are hyperinsulinemia, hyperglycemia, hyperlipidemia, increased inflammatory and reduced insulin response. Aspirin (ASA and other non-steroidal anti-inflammatory drugs (NSAIDs have been associated with the prevention of diabetes, obesity and related cardiovascular disorders. Aspirin has been used in many clinical and experimental trials for the prevention of diabetes and associated complications. Methods: In this study, five month old Goto-Kakizaki (GK rats, which showed signs of mild hyperglycemia (fasting blood glucose 80-95 mg/dl vs 55-60 mg/dl Wistar control rats were used. Two subgroups of GK and Wistar control rats were injected intraperitoneally with 100 mg aspirin/kg body weight/ day for 5 weeks. Animals were sacrificed and blood and tissues were collected after performing glucose tolerance (2 h post 2g IP glucose ingestion tests in experimental and control groups. Results: Aspirin caused a moderate decrease in hyperglycemia. However, we observed a significant improvement in glucose tolerance after ASA treatment in GK rats compared to the nondiabetic Wistar rats. Also, the ASA treated GK rats exhibited a significant decrease in insulinemia. ASA treatment also caused a marked reduction in the pro-inflammatory prostaglandin, PGE2, which was significantly higher in GK rats. On the other hand, no significant organ toxicity was observed after ASA treatment at this dose and time period. However, the total cholesterol and lipoprotein levels were significantly increased in GK rats, which decreased after ASA treatment. Immunofluorescence staining for insulin/glucagon secreting pancreatic cells showed improved beta-cell structural and functional integrity in ASA-treated rats which was also confirmed by SDS-PAGE and Western blot analysis

Phosphorylation events implicating p38 and PI3K mediate tungstate-effects in MIN6 beta cells

International Nuclear Information System (INIS)

Piquer, Sandra; Barcelo-Batllori, Silvia; Julia, Marta; Marzo, Nuria; Nadal, Belen; Guinovart, Joan J.; Gomis, Ramon

2007-01-01

Oral administration of sodium tungstate is an effective treatment for diabetes in animal models. Several lines of evidence indicate the pancreatic beta cell as one of the targets of tungstate action. Here, we examined the molecular mechanism by which this compound exerts its effects on the beta cell line MIN6. Tungstate treatment induced phosphorylation and subsequent activation of p38 and PI3K which in turn are implicated in tungstate PDX-1 nuclear localization and activation. Although no effect was observed in glucose-induced insulin secretion we found that tungstate activates basal insulin release, a process driven, at least in part, by activation of p38. These results show a direct involvement of p38 and PI3K phosphorylation in the mechanism of action of tungstate in the beta cell

In vivo and in vitro characterization of [{sup 18}F]-FE-(+)-DTBZ as a tracer for beta-cell mass

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Eriksson, Olof [Division of Radiology, Department of Oncology, Radiology and Clinical Immunology, University Hospital, 751 87 Uppsala (Sweden)], E-mail: olof.eriksson@radiol.uu.se; Jahan, Mahabuba [Department of Clinical Neuroscience, Karolinska Institute, Section of Psychiatry, Karolinska Hospital, 171 76, Stockholm (Sweden); Johnstroem, Peter [Department of Clinical Neuroscience, Karolinska Institute, Section of Psychiatry, Karolinska Hospital, 171 76, Stockholm (Sweden); CNS and Pain Control, AstraZeneca R and D, 151 36, Soedertaelje (Sweden); Korsgren, Olle [Department of Oncology, Radiology and Clinical Immunology, Division of Clinical Immunology, University Hospital, 751 87 Uppsala (Sweden); Sundin, Anders [Division of Radiology, Department of Oncology, Radiology and Clinical Immunology, University Hospital, 751 87 Uppsala (Sweden); Department of Radiology, Karolinska University Hospital, 171 76, Stockholm (Sweden); Halldin, Christer [Department of Clinical Neuroscience, Karolinska Institute, Section of Psychiatry, Karolinska Hospital, 171 76, Stockholm (Sweden); Johansson, Lars [Division of Radiology, Department of Oncology, Radiology and Clinical Immunology, University Hospital, 751 87 Uppsala (Sweden); AstraZeneca R and D, 431 50, Moelndal (Sweden)

2010-04-15

Introduction: The positron emission tomography (PET) tracer 9-[{sup 18}F]fluoroethyl-(+)-dihydrotetrabenazine ([{sup 18}F]-FE-(+)-DTBZ) is a potential candidate for quantifying beta-cell mass in vivo. The purpose was to investigate in vitro and in vivo utility of this tracer for the assessment of beta-cell mass. Methods: Three pigs were intravenously administered [{sup 18}F]-FE-(+)-DTBZ and examined by PET/computed tomography. Binding parameters were estimated by kinetic modeling. In vitro k{sub D} and B{sub max} were determined by saturation binding studies of endocrine and exocrine human tissue homogenates. In vitro pancreatic uptake was determined by tissue autoradiography with pancreases from patients with types 1 (T1DM) and 2 diabetes mellitus (T2DM) and healthy controls. Results: [{sup 18}F]-FE-(+)-DTBZ had a k{sub D} of 3.5{+-}1.0 nM, a B{sub max} of 382{+-}108 fmol/mg protein and a specificity of 89{+-}1.8% in islet homogenates. The total exocrine uptake was lower and 65% was nondisplaceable. No uptake difference was observed in pancreatic tissue slices from patients with T1DM, T2DM or healthy controls. The in vivo porcine pancreatic uptake reached a peak of standardized uptake value (SUV) of 2.8 with a low distribution volume ratio in all animals. Moderate to high tracer uptake was identified in the bile system and in bone. Conclusions: [{sup 18}F]-FE-(+)-DTBZ binds to vesicular monoamine transporter 2 (VMAT2) with high specificity in pure islet tissue in vitro. However, there is high nondisplaceable binding to exocrine tissue. In addition, in vivo tracer metabolism and dehalogenation result in severe underestimation of porcine pancreatic VMAT2 expression and BCM. The results do not support [{sup 18}F]-FE-(+)-DTBZ as a suitable tracer for in vivo beta-cell imaging.

Do immunotherapy and beta cell replacement play a synergistic role in the treatment of type 1 diabetes?

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Li, Dong-Sheng; Warnock, Garth L; Tu, Han-Jun; Ao, Ziliang; He, Zehua; Lu, Hong; Dai, Long-Jun

2009-10-07

Type 1 diabetes (T1D) is the result of the autoimmune response against pancreatic insulin-producing ss-cells. Its ultimate consequence is beta-cell insufficiency-mediated dysregulation of blood glucose control. In terms of T1D treatment, immunotherapy addresses the cause of T1D, mainly through re-setting the balance between autoimmunity and regulatory mechanisms. Regulatory T cells play an important role in this immune intervention. An alternative T1D treatment is beta-cell replacement, which can reverse the consequence of the disease by replacing destroyed beta-cells in the diabetic pancreas. The applicable insulin-producing cells can be directly obtained from islet transplantation or generated from other cell sources such as autologous adult stem cells, embryonic stem cells, and induced pluripotent stem cells. In this review, we summarize the recent research progress and analyze the possible advantages and disadvantages of these two therapeutic options especially focusing on the potential synergistic effect on T1D treatment. Exploring the optimal combination of immunotherapy and beta-cell replacement will pave the way to the most effective cure for this devastating disease.

Beta cell adaptation in pregnancy

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Nielsen, Jens Høiriis

2016-01-01

Pregnancy is associated with a compensatory increase in beta cell mass. It is well established that somatolactogenic hormones contribute to the expansion both indirectly by their insulin antagonistic effects and directly by their mitogenic effects on the beta cells via receptors for prolactin...... and growth hormone expressed in rodent beta cells. However, the beta cell expansion in human pregnancy seems to occur by neogenesis of beta cells from putative progenitor cells rather than by proliferation of existing beta cells. Claes Hellerström has pioneered the research on beta cell growth for decades...... in the expansion of the beta cell mass in human pregnancy, and the relative roles of endocrine factors and nutrients....

Lipotoxicity induces hepatic protein inclusions through TBK1-mediated p62/SQSTM1 phosphorylation.

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Cho, Chun-Seok; Park, Hwan-Woo; Ho, Allison; Semple, Ian A; Kim, Boyoung; Jang, Insook; Park, Haeli; Reilly, Shannon; Saltiel, Alan R; Lee, Jun Hee

2017-12-18

Obesity commonly leads to hepatic steatosis, which often provokes lipotoxic injuries to hepatocytes that cause non-alcoholic steatohepatitis (NASH). NASH in turn is associated with the accumulation of insoluble protein aggregates that are composed of ubiquitinated proteins and ubiquitin adaptor p62/sequestosome 1 (SQSTM1). The formation of p62 inclusions in hepatocytes is the critical marker that distinguishes simple fatty liver from NASH and predicts a poor prognostic outcome for subsequent liver carcinogenesis. However, the molecular mechanism by which lipotoxicity induces protein aggregation is currently unknown. Here we show that upon saturated fatty acid-induced lipotoxicity, Tank-binding protein kinase 1 (TBK1) is activated and phosphorylates p62. The TBK1-mediated p62 phosphorylation is important for lipotoxicity-induced aggregation of ubiquitinated proteins and the formation of large protein inclusions in hepatocytes. In addition, cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING), upstream regulators of TBK1, are involved in the lipotoxic activation of TBK1 and subsequent p62 phosphorylation in hepatocytes. Furthermore, TBK1 inhibition prevented formation of the ubiquitin-p62 aggregates, not only in cultured hepatocytes, but also in mouse models of obesity and NASH. These results suggest that lipotoxic activation of TBK1 and subsequent p62 phosphorylation are critical steps in the NASH pathology of protein inclusion accumulation in hepatocytes. This mechanism can provide an explanation for how hypernutrition and obesity promote the development of severe liver pathologies, such as steatohepatitis and liver cancer, by facilitating the formation of p62 inclusions. This article is protected by copyright. All rights reserved. © 2017 by the American Association for the Study of Liver Diseases.

Regulation of beta cell replication

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Lee, Ying C; Nielsen, Jens Høiriis

2008-01-01

Beta cell mass, at any given time, is governed by cell differentiation, neogenesis, increased or decreased cell size (cell hypertrophy or atrophy), cell death (apoptosis), and beta cell proliferation. Nutrients, hormones and growth factors coupled with their signalling intermediates have been...... suggested to play a role in beta cell mass regulation. In addition, genetic mouse model studies have indicated that cyclins and cyclin-dependent kinases that determine cell cycle progression are involved in beta cell replication, and more recently, menin in association with cyclin-dependent kinase...... inhibitors has been demonstrated to be important in beta cell growth. In this review, we consider and highlight some aspects of cell cycle regulation in relation to beta cell replication. The role of cell cycle regulation in beta cell replication is mostly from studies in rodent models, but whether...

Islet Cells Serve as Cells of Origin of Pancreatic Gastrin-Positive Endocrine Tumors

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Bonnavion, Rémy; Teinturier, Romain; Jaafar, Rami

2015-01-01

The cells of origin of pancreatic gastrinomas remain an enigma, since no gastrin-expressing cells are found in the normal adult pancreas. It was proposed that the cellular origin of pancreatic gastrinomas may come from either the pancreatic cells themselves or gastrin-expressing cells which have ...

Immune-mediated beta-cell destruction in vitro and in vivo-A pivotal role for galectin-3

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Karlsen, Allan E; Størling, Zenia M; Sparre, Thomas

2006-01-01

Pro-apoptotic cytokines are toxic to the pancreatic beta-cells and have been associated with the pathogenesis of Type 1 diabetes (T1D). Proteome analysis of IL-1beta exposed isolated rat islets identified galectin-3 (gal-3) as the most up-regulated protein. Here analysis of human and rat islets a....... In summary, combined proteome-transcriptome-genome and functional analyses identify gal-3 as a candidate gene/protein in T1D susceptibility that may prove valuable in future intervention/prevention strategies....

Susceptibility of ATM-deficient pancreatic cancer cells to radiation.

Science.gov (United States)

Ayars, Michael; Eshleman, James; Goggins, Michael

2017-05-19

Ataxia telangiectasia mutated (ATM) is inactivated in a significant minority of pancreatic ductal adenocarcinomas and may be predictor of treatment response. We determined if ATM deficiency renders pancreatic cancer cells more sensitive to fractionated radiation or commonly used chemotherapeutics. ATM expression was knocked down in three pancreatic cancer cell lines using ATM-targeting shRNA. Isogenic cell lines were tested for sensitivity to several chemotherapeutic agents and radiation. DNA repair kinetics were analyzed in irradiated cells using the comet assay. We find that while rendering pancreatic cancer cells ATM-deficient did not significantly change their sensitivity to several chemotherapeutics, it did render them exquisitely sensitized to radiation. Pancreatic cancer ATM status may help predict response to radiotherapy.

Specifying pancreatic endocrine cell fates.

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Collombat, Patrick; Hecksher-Sørensen, Jacob; Serup, Palle; Mansouri, Ahmed

2006-07-01

Cell replacement therapy could represent an attractive alternative to insulin injections for the treatment of diabetes. However, this approach requires a thorough understanding of the molecular switches controlling the specification of the different pancreatic cell-types in vivo. These are derived from an apparently identical pool of cells originating from the early gut endoderm, which are successively specified towards the pancreatic, endocrine, and hormone-expressing cell lineages. Numerous studies have outlined the crucial roles exerted by transcription factors in promoting the cell destiny, defining the cell identity and maintaining a particular cell fate. This review focuses on the mechanisms regulating the morphogenesis of the pancreas with particular emphasis on recent findings concerning the transcription factor hierarchy orchestrating endocrine cell fate allocation.

α-Ketoglutarate Promotes Pancreatic Progenitor-Like Cell Proliferation

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Jing Song

2018-03-01

Full Text Available A major source of β cell generation is pancreatic progenitor-like cell differentiation. Multiple studies have confirmed that stem cell metabolism plays important roles in self-renewal and proliferation. In the absence of glucose, glutamine provides the energy for cell division and growth. Furthermore, α-ketoglutarate (αKG, a precursor for glutamine synthesis, is sufficient for enabling glutamine-independent cell proliferation. We have demonstrated that αKG contributes to the large-scale proliferation of pancreatic progenitor-like cells that can provide an ample amount of clinically relevant β cells. We compared the mRNA expression of a subset of genes, the abundance of ATP, reactive oxide species, mitochondrial number, and the colony-forming frequency between mouse pancreatic CD133+ and CD133− cells. We employed Real-Time PCR, immunostaining and passage assays to investigate self-renewal and proliferation of pancreatic progenitor-like cells in a 3D culture system in the presence and absence of αKG. The energy metabolism of CD133+ cells was more prone to oxidative phosphorylation. However, in the 3D culture system, when αKG was supplemented to the culture medium, the proliferation of the pancreatic progenitor-like cells was significantly elevated. We confirmed that the presence of αKG correlated with the up-regulation of Ten-Eleven Translocation (Tet. αKG can promote the proliferation of pancreatic progenitor-like cells via the up-regulation of Tet.

α-Ketoglutarate Promotes Pancreatic Progenitor-Like Cell Proliferation.

Science.gov (United States)

Song, Jing; Ma, Dongshen; Xing, Yun; Tang, Shanshan; Alahdal, Murad; Guo, Jiamin; Pan, Yi; Zhang, Yanfeng; Shen, Yumeng; Wu, Qiong; Lu, Zhou; Jin, Liang

2018-03-22

A major source of β cell generation is pancreatic progenitor-like cell differentiation. Multiple studies have confirmed that stem cell metabolism plays important roles in self-renewal and proliferation. In the absence of glucose, glutamine provides the energy for cell division and growth. Furthermore, α-ketoglutarate (αKG), a precursor for glutamine synthesis, is sufficient for enabling glutamine-independent cell proliferation. We have demonstrated that αKG contributes to the large-scale proliferation of pancreatic progenitor-like cells that can provide an ample amount of clinically relevant β cells. We compared the mRNA expression of a subset of genes, the abundance of ATP, reactive oxide species, mitochondrial number, and the colony-forming frequency between mouse pancreatic CD133⁺ and CD133 - cells. We employed Real-Time PCR, immunostaining and passage assays to investigate self-renewal and proliferation of pancreatic progenitor-like cells in a 3D culture system in the presence and absence of αKG. The energy metabolism of CD133⁺ cells was more prone to oxidative phosphorylation. However, in the 3D culture system, when αKG was supplemented to the culture medium, the proliferation of the pancreatic progenitor-like cells was significantly elevated. We confirmed that the presence of αKG correlated with the up-regulation of Ten-Eleven Translocation (Tet). αKG can promote the proliferation of pancreatic progenitor-like cells via the up-regulation of Tet.

The functional performance of microencapsulated human pancreatic islet-derived precursor cells.

Science.gov (United States)

Montanucci, Pia; Pennoni, Ilaria; Pescara, Teresa; Blasi, Paolo; Bistoni, Giovanni; Basta, Giuseppe; Calafiore, Riccardo

2011-12-01

We have examined long-term cultured, human islet-derived stem/precursor cells (hIPC). Whole human islets (HI) were obtained by multi-enzymatic digestion of cadaveric donor pancreases, plated on tissue flasks, and allowed to adhere and expand for several in vitro passages, in order to obtain hIPC. We detected specific stem cell markers (Oct-4, Sox-2, Nanog, ABCG2, Klf-4, CD117) in both intact HI and hIPC. Moreover, hIPC while retaining the expression of Glut-2, Pdx-1, CK-19, and ICA-512, started re-expressing Ngn3, thereby indicating acquisition of a specific pancreatic islet beta cell-oriented phenotype identity. The intrinsic plasticity of hIPC was documented by their ability to differentiate into various germ layer-derived cell phenotypes (ie, osteocytic, adipocytic and neural), including endocrine cells associated with insulin secretory capacity. To render hIPC suitable for transplantation we have enveloped them within our highly purified, alginate-based microcapsules. Upon intraperitoneal graft in NOD/SCID mice we have observed that the microcapsules acted as three-dimensional niches favouring post-transplant hIPC differentiation and acquisition of beta cell-like functional competence. Copyright © 2011 Elsevier Ltd. All rights reserved.

Divergent Effects of Dendritic Cells on Pancreatitis

Science.gov (United States)

2015-09-01

role of dendritic cells in pancreatitis. Dendritic cells are professional antigen presenting cells which initiate innate and adaptive immune... Lymphoid -tissue-specific homing of bone- marrow-derived dendritic cells . Blood. 113:6638–6647. http://dx.doi .org/10.1182/blood-2009-02-204321 Dapito...Award Number: W81XWH-12-1-0313 TITLE: Divergent Effects of Dendritic Cells on Pancreatitis PRINCIPAL INVESTIGATOR: Dr. George Miller

Mesenchymal Stromal Cell Therapy for Pancreatitis: A Systematic Review

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Sara M. Ahmed

2018-01-01

Full Text Available Background. Based on animal studies, adult mesenchymal stromal cells (MSCs are promising for the treatment of pancreatitis. However, the best type of this form of cell therapy and its mechanism of action remain unclear. Methods. We searched the PubMed, Web of Science, Scopus, Google Scholar, and Clinical Trials.gov websites for studies using MSCs as a therapy for both acute and chronic pancreatitis published until September 2017. Results. We identified 276 publications; of these publications, 18 met our inclusion criteria. In animal studies, stem cell therapy was applied more frequently for acute pancreatitis than for chronic pancreatitis. No clinical trials were identified. MSC therapy ameliorated pancreatic inflammation in acute pancreatitis and pancreatic fibrosis in chronic pancreatitis. Bone marrow and umbilical cord MSCs were the most frequently administered cell types. Due to the substantial heterogeneity among the studies regarding the type, source, and dose of MSCs used, conducting a meta-analysis was not feasible to determine the best type of MSCs. Conclusion. The available data were insufficient for determining the best type of MSCs for the treatment of acute or chronic pancreatitis; therefore, clinical trials investigating the use of MSCs as therapy for pancreatitis are not warranted.

Characterization of Phospholipids in Insulin Secretory Granules and Mitochondria in Pancreatic Beta Cells and Their Changes with Glucose Stimulation*

Science.gov (United States)

MacDonald, Michael J.; Ade, Lacmbouh; Ntambi, James M.; Ansari, Israr-Ul H.; Stoker, Scott W.

2015-01-01

The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. PMID:25762724

Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

International Nuclear Information System (INIS)

Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

2015-01-01

Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

Energy Technology Data Exchange (ETDEWEB)

Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng, E-mail: oxyccc@163.com

2015-12-04

Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells.

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Mohammad Massumi

Full Text Available The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have developed an abbreviated five-stage protocol (25-30 days to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs. We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP, SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL, and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion, targeting selected signaling pathways for 25-30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs, small molecules or genes that may have potential to influence beta-cell function.

ENDOCRINE PANCREATIC FUNCTION IN ACUTE PANCREATITIS

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P. V. Novokhatny

2014-02-01

Full Text Available Introduction Among the organs of internal secretion pancreas has a special place thanks to active exocrine function and a wide range of physiological actions of produced hormones. Violations of endocrine pancreas arises in 6.5-38 % of patients with acute pancreatitis. However, there is still no clear understanding of the pathogenetic mechanisms of hormonal dysfunction of the pancreas in acute pancreatitis, there is no uniform algorithms for its correction. Aim of the research was to study the endocrine function of pancreas in acute pancreatitis. To define the role of endocrine pancreatic function in the etiology and pathogenesis of the acute pancreatitis. To assess the prospects of the use of pancreatic hormones in the treatment and predicting the outcomes of acute pancreatitis. Materials and methods of the research Survey of publications in specialized periodical medical journals, PubMed sources developed by the National Center for Biotechnology Information. Search in PubMed was carried out in the following databases: MEDLINE, Pre MEDLINE. Results of the research. In a significant proportion of patients who recovered from acute pancreatitis, exocrine and endocrine functional impairments were found. This finding was not detected only in patients after severe acute pancreatitis. Routine evaluation of pancreatic function after acute pancreatitis should be considered. The comparative analysis of the synthetic analogues (somatostatin, calcitonin, leu-enkefalin-dalargin influence on the glucose metabolism of rats in acute pancreatitis of was made. Physiological reaction of beta-cells is preserved in infusion of somatostatin. However, infusion of calcitonin results in the distortion of counterregulatory action of insulin and glucagon. It was detected that pancreatic renin-angiotensin system is markedly activated in the experimental rat models of chronic hypoxia and acute pancreatitis. The activation of the pancreatic renin-angiotensin system by

Beta Lactamase Producing Clostridium perfringens Bacteremia in an Elderly Man with Acute Pancreatitis

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Rashmi Mishra

2016-01-01

Full Text Available Clostridium perfringens bacteremia is associated with adverse outcomes. Known risk factors include chronic kidney disease, malignancy, diabetes mellitus, and gastrointestinal disease. We present a 74-year-old man admitted with confusion, vomiting, and abdominal pain. Exam revealed tachycardia, hypotension, lethargy, distended abdomen, and cold extremities. He required intubation and aggressive resuscitation for septic shock. Laboratory data showed leukocytosis, metabolic acidosis, acute kidney injury, and elevated lipase. CT scan of abdomen revealed acute pancreatitis and small bowel ileus. He was started on vancomycin and piperacillin-tazobactam. Initial blood cultures were positive for C. perfringens on day five. Metronidazole and clindamycin were added to the regimen. Repeat CT (day 7 revealed pancreatic necrosis. The patient developed profound circulatory shock requiring multiple vasopressors, renal failure requiring dialysis, and bacteremia with vancomycin-resistant enterococci. Hemodynamic instability precluded surgical intervention and he succumbed to multiorgan failure. Interestingly, our isolate was beta lactamase producing. We review the epidemiology, risk factors, presentation, and management of C. perfringens bacteremia. This case indicates a need for high clinical suspicion for clostridial sepsis and that extended spectrum beta lactam antibiotic coverage may be inadequate and should be supplemented with use of clindamycin or metronidazole if culture is positive, until sensitivities are known.

Reference intervals for glucose, beta-cell polypeptides and counterregulatory factors during prolonged fasting

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Højlund, Kurt; Wildner-Christensen, M; Eshøj, O

2001-01-01

To establish reference intervals for the pancreatic beta-cell response and the counterregulatory hormone response to prolonged fasting, we studied 33 healthy subjects (16 males, 17 females) during a 72-h fast. Glucose, insulin, C-peptide, and proinsulin levels decreased (P ... of counterregulatory factors increased during the fast [P fasting (P ... decreased from the second to third day of fasting (P = 0.03). Males had higher glucose and glucagon levels and lower FFA levels during the fast (P

Expression of transforming growth factor beta (TGF beta) receptors and expression of TGF beta 1, TGF beta 2 and TGF beta 3 in human small cell lung cancer cell lines

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Damstrup, L; Rygaard, K; Spang-Thomsen, M

1993-01-01

A panel of 21 small cell lung cancer cell (SCLC) lines were examined for the presence of Transforming growth factor beta receptors (TGF beta-r) and the expression of TGF beta mRNAs. By the radioreceptor assay we found high affinity receptors to be expressed in six cell lines. scatchard analysis......(r) = 65,000 and 90,000 and the betaglycan (type III) with M(r) = 280,000. Northern blotting showed expression of TGF beta 1 mRNA in ten, TGF beta 2 mRNA in two and TGF beta 3 mRNA in seven cell lines. Our results provide, for the first time, evidence that a large proportion of a broad panel of SCLC cell...... lines express TGF beta-receptors and also produce TGF beta mRNAs....

Pancreatic β-cell regeneration: Facultative or dedicated progenitors?

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Afelik, Solomon; Rovira, Meritxell

2017-04-15

The adult pancreas is only capable of limited regeneration. Unlike highly regenerative tissues such as the skin, intestinal crypts and hematopoietic system, no dedicated adult stem cells or stem cell niche have so far been identified within the adult pancreas. New β cells have been shown to form in the adult pancreas, in response to high physiological demand or experimental β-cell ablation, mostly by replication of existing β cells. The possibility that new β cells are formed from other sources is currently a point of major controversy. Under particular injury conditions, fully differentiated pancreatic duct and acinar cells have been shown to dedifferentiate into a progenitor-like state, however the extent, to which ductal, acinar or other endocrine cells contribute to restoring pancreatic β-cell mass remains to be resolved. In this review we focus on regenerative events in the pancreas with emphasis on the restoration of β-cell mass. We present an overview of regenerative responses noted within the different pancreatic lineages, following injury. We also highlight the intrinsic plasticity of the adult pancreas that allows for inter-conversion of fully differentiated pancreatic lineages through manipulation of few genes or growth factors. Taken together, evidence from a number of studies suggest that differentiated pancreatic lineages could act as facultative progenitor cells, but the extent to which these contribute to β-cell regeneration in vivo is still a matter of contention. Copyright © 2016. Published by Elsevier B.V.

FGF7 and cell density are required for final differentiation of pancreatic amylase-positive cells from human ES cells.

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Takizawa-Shirasawa, Sakiko; Yoshie, Susumu; Yue, Fengming; Mogi, Akimi; Yokoyama, Tadayuki; Tomotsune, Daihachiro; Sasaki, Katsunori

2013-12-01

The major molecular signals of pancreatic exocrine development are largely unknown. We examine the role of fibroblast growth factor 7 (FGF7) in the final induction of pancreatic amylase-containing exocrine cells from induced-pancreatic progenitor cells derived from human embryonic stem (hES) cells. Our protocol consisted in three steps: Step I, differentiation of definitive endoderm (DE) by activin A treatment of hES cell colonies; Step II, differentiation of pancreatic progenitor cells by re-plating of the cells of Step I onto 24-well plates at high density and stimulation with all-trans retinoic acid; Step III, differentiation of pancreatic exocrine cells with a combination of FGF7, glucagon-like peptide 1 and nicotinamide. The expression levels of pancreatic endodermal markers such as Foxa2, Sox17 and gut tube endoderm marker HNF1β were up-regulated in both Step I and II. Moreover, in Step III, the induced cells expressed pancreatic markers such as amylase, carboxypeptidase A and chymotrypsinogen B, which were similar to those in normal human pancreas. From day 8 in Step III, cells immunohistochemically positive for amylase and for carboxypeptidase A, a pancreatic exocrine cell product, were induced by FGF7. Pancreatic progenitor Pdx1-positive cells were localized in proximity to the amylase-positive cells. In the absence of FGF7, few amylase-positive cells were identified. Thus, our three-step culture protocol for human ES cells effectively induces the differentiation of amylase- and carboxypeptidase-A-containing pancreatic exocrine cells.

Ceramide-Protein Interactions Modulate Ceramide-Associated Lipotoxic Cardiomyopathy

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Stanley M. Walls

2018-03-01

Full Text Available Lipotoxic cardiomyopathy (LCM is characterized by abnormal myocardial accumulation of lipids, including ceramide; however, the contribution of ceramide to the etiology of LCM is unclear. Here, we investigated the association of ceramide metabolism and ceramide-interacting proteins (CIPs in LCM in the Drosophila heart model. We find that ceramide feeding or ceramide-elevating genetic manipulations are strongly associated with cardiac dilation and defects in contractility. High ceramide-associated LCM is prevented by inhibiting ceramide synthesis, establishing a robust model of direct ceramide-associated LCM, corroborating previous indirect evidence in mammals. We identified several CIPs from mouse heart and Drosophila extracts, including caspase activator Annexin-X, myosin chaperone Unc-45, and lipogenic enzyme FASN1, and remarkably, their cardiac-specific manipulation can prevent LCM. Collectively, these data suggest that high ceramide-associated lipotoxicity is mediated, in part, through altering caspase activation, sarcomeric maintenance, and lipogenesis, thus providing evidence for conserved mechanisms in LCM pathogenesis in mammals.

Characterization of stimulus-secretion coupling in the human pancreatic EndoC-βH1 beta cell line.

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Lotta E Andersson

Full Text Available Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay, gene expression (Gene Chip array, metabolite levels (GC/MS, respiration (Seahorse XF24 Extracellular Flux Analyzer, glucose utilization (radiometric, lactate release (enzymatic colorimetric, ATP levels (enzymatic bioluminescence and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the

miR-146a Suppresses Invasion of Pancreatic Cancer Cells

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Li, Yiwei; VandenBoom, Timothy G.; Wang, Zhiwei; Kong, Dejuan; Ali, Shadan; Philip, Philip A.; Sarkar, Fazlul H.

2010-01-01

The aggressive course of pancreatic cancer is believed to reflect its unusually invasive and metastatic nature, which is associated with epidermal growth factor receptor (EGFR) overexpression and NF-κB activation. MicroRNAs (miRNA) have been implicated in the regulation of various pathobiological processes in cancer, including metastasis in pancreatic cancer and in other human malignancies. In this study, we report lower expression of miR-146a in pancreatic cancer cells compared with normal human pancreatic duct epithelial cells. Reexpression of miR-146a inhibited the invasive capacity of pancreatic cancer cells with concomitant downregulation of EGFR and the NF-κB regulatory kinase interleukin 1 receptor–associated kinase 1 (IRAK-1). Cellular mechanism studies revealed crosstalk between EGFR, IRAK-1, IκBα, NF-κB, and MTA-2, a transcription factor that regulates metastasis. Treatment of pancreatic cancer cells with the natural products 3,3′-diinodolylmethane (DIM) or isoflavone, which increased miR-146a expression, caused a downregulation of EGFR, MTA-2, IRAK-1, and NF-κB, resulting in an inhibition of pancreatic cancer cell invasion. Our findings reveal DIM and isoflavone as nontoxic activators of a miRNA that can block pancreatic cancer cell invasion and metastasis, offering starting points to design novel anticancer agents. PMID:20124483

Stem cell-based approach in diabetes and pancreatic cancer management

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Yi-Zhou Jiang

2017-01-01

Full Text Available Stem cell-mediated therapy is a promising strategy for treating pancreatic diseases such as Type-1 diabetes (T1D and pancreatic cancers. Although islet transplantation has been reported to be an effective diabetes therapy, its worldwide application is extremely limited due to the shortage of donor islets and immune rejection problems. Stem cell-based approach for islet neogenesis in vivo could provide a promising alternative source of islets for treating diabetes. On the other hand, targeting the cancer stem cells could be very effective for the treatment of pancreatic cancers. In this review, we focused on the present progress in the field of adult pancreatic stem cells, stem cell-mediated strategies for treating T1D, and pancreatic cancer stem cells, while discussing of the possible challenges involved in them.

Effects of the hypoglycaemic drugs repaglinide and glibenclamide on ATP-sensitive potassium-channels and cytosolic calcium levels in beta TC3 cells and rat pancreatic beta cells

DEFF Research Database (Denmark)

Gromada, J; Dissing, S; Kofod, Hans

1995-01-01

The present study demonstrates the action of the hypoglycaemic drugs repaglinide and glibenclamide in cultured newborn rat islet cells and mouse beta TC3 cells. In cell-attached membrane patches of newborn rat islet cells repaglinide (10 nmol/l) and glibenclamide (20 nmol/l) decrease the open pro...

Characterization of phospholipids in insulin secretory granules and mitochondria in pancreatic beta cells and their changes with glucose stimulation.

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MacDonald, Michael J; Ade, Lacmbouh; Ntambi, James M; Ansari, Israr-Ul H; Stoker, Scott W

2015-04-24

The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Echovirus 6 Infects Human Exocrine and Endocrine Pancreatic Cells and Induces Pro-Inflammatory Innate Immune Response

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Luis Sarmiento

2017-01-01

Full Text Available Human enteroviruses (HEV, especially coxsackievirus serotype B (CVB and echovirus (E, have been associated with diseases of both the exocrine and endocrine pancreas, but so far evidence on HEV infection in human pancreas has been reported only in islets and ductal cells. This study aimed to investigate the capability of echovirus strains to infect human exocrine and endocrine pancreatic cells. Infection of explanted human islets and exocrine cells with seven field strains of E6 caused cytopathic effect, virus titer increase and production of HEV protein VP1 in both cell types. Virus particles were found in islets and acinar cells infected with E6. No cytopathic effect or infectious progeny production was observed in exocrine cells exposed to the beta cell-tropic strains of E16 and E30. Endocrine cells responded to E6, E16 and E30 by upregulating the transcription of interferon-induced with helicase C domain 1 (IF1H1, 2'-5'-oligoadenylate synthetase 1 (OAS1, interferon-β (IFN-β, chemokine (C–X–C motif ligand 10 (CXCL10 and chemokine (C–C motif ligand 5 (CCL5. Echovirus 6, but not E16 or E30, led to increased transcription of these genes in exocrine cells. These data demonstrate for the first time that human exocrine cells represent a target for E6 infection and suggest that certain HEV serotypes can replicate in human pancreatic exocrine cells, while the pancreatic endocrine cells are permissive to a wider range of HEV.

Tissue-specific methylation of human insulin gene and PCR assay for monitoring beta cell death.

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Mohamed I Husseiny

Full Text Available The onset of metabolic dysregulation in type 1 diabetes (T1D occurs after autoimmune destruction of the majority of pancreatic insulin-producing beta cells. We previously demonstrated that the DNA encoding the insulin gene is uniquely unmethylated in these cells and then developed a methylation-specific PCR (MSP assay to identify circulating beta cell DNA in streptozotocin-treated mice prior to the rise in blood glucose. The current study extends to autoimmune non-obese diabetic (NOD mice and humans, showing in NOD mice that beta cell death occurs six weeks before the rise in blood sugar and coincides with the onset of islet infiltration by immune cells, demonstrating the utility of MSP for monitoring T1D. We previously reported unique patterns of methylation of the human insulin gene, and now extend this to other human tissues. The methylation patterns of the human insulin promoter, intron 1, exon 2, and intron 2 were determined in several normal human tissues. Similar to our previous report, the human insulin promoter was unmethylated in beta cells, but methylated in all other tissues tested. In contrast, intron 1, exon 2 and intron 2 did not exhibit any tissue-specific DNA methylation pattern. Subsequently, a human MSP assay was developed based on the methylation pattern of the insulin promoter and human islet DNA was successfully detected in circulation of T1D patients after islet transplantation therapy. Signal levels of normal controls and pre-transplant samples were shown to be similar, but increased dramatically after islet transplantation. In plasma the signal declines with time but in whole blood remains elevated for at least two weeks, indicating that association of beta cell DNA with blood cells prolongs the signal. This assay provides an effective method to monitor beta cell destruction in early T1D and in islet transplantation therapy.

Pancreatic iron stores assessed by magnetic resonance imaging (MRI) in beta thalassemic patients

International Nuclear Information System (INIS)

Feitosa de Assis, Reijâne Alves; Ribeiro, Andreza Alice; Kay, Fernando Uliana; Rosemberg, Laércio Alberto; Nomura, Cesar Higa; Loggetto, Sandra Regina; Araujo, Aderson S.; Fabron Junior, Antonio; Pinheiro de Almeida Veríssimo, Mônica; Baldanzi, Giorgio Roberto; Espósito, Breno Pannia; Baroni, Ronaldo Hueb; Wood, John C.; Hamerschlak, Nelson

2012-01-01

Purpose: To assess the correlation between MRI findings of the pancreas with those of the heart and liver in patients with beta thalassemia; to compare the pancreas T2* MRI results with glucose and ferritin levels and labile plasma iron (LPI). Materials and methods: We retrospectively evaluated chronically transfused patients, testing glucose with enzymatic tests, serum ferritin with chemiluminescence, LPI with cellular fluorescence, and T2* MRI to assess iron content in the heart, liver, and pancreas. MRI results were compared with one another and with serum glucose, ferritin, and LPI. Liver iron concentration (LIC) was determined in 11 patients’ liver biopsies by atomic absorption spectrometry. Results: 289 MRI studies were available from 115 patients during the period studied. 9.4% of patients had overt diabetes and an additional 16% of patients had impaired fasting glucose. Both pancreatic and cardiac R2* had predictive power (p 2 = 0.20, p 2 = 0.09, p 2 = 0.14, p 2 = 0.03, p 2 of 0.91. Conclusions: Pancreatic iron overload can be assessed by MRI, but siderosis in other organs did not correlate significantly with pancreatic hemosiderosis.

ATP release, generation and hydrolysis in exocrine pancreatic duct cells

DEFF Research Database (Denmark)

Kowal, Justyna Magdalena; Yegutkin, G.G.; Novak, Ivana

2015-01-01

Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, ou...... may be important in pancreas physiology and potentially in pancreas pathophysiology....... aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan...

Computed tomography findings of pancreatic metastases from renal cell carcinoma

International Nuclear Information System (INIS)

Prando, Adilson

2008-01-01

Objective: To present computed tomography findings observed in four patients submitted to radical nephrectomy for renal cell carcinoma who developed pancreatic metastases afterwards. Materials and methods: The four patients underwent radical nephrectomy for stage Tz1 (n=2) and stage T3a (n=2) renal cell carcinoma. The mean interval between nephrectomy and detection of pancreatic metastases was eight years. Two asymptomatic patients presented with solitary pancreatic metastases (confined to the pancreas). Two symptomatic patients presented with single and multiple pancreatic metastases, both with tumor recurrence in the contralateral kidney. Results: Computed tomography studies demonstrated pancreatic metastases as solitary (n=2), single (n=1) or multiple (n=1) hypervascular lesions. Partial pancreatectomy was performed in two patients with solitary pancreatic metastases and both are free of disease at four and two years after surgery. Conclusion: Pancreatic metastases from renal cell carcinoma are rare and can occur many years after the primary tumor presentation. Multiple pancreatic metastases and pancreatic metastases associated with tumor recurrence in the contralateral kidney are uncommon. Usually, on computed tomography images pancreatic metastases are visualized as solitary hypervascular lesions, simulating isletcell tumors. Surgical management should be considered for patients with solitary pancreatic lesions. (author)

Computed tomography findings of pancreatic metastases from renal cell carcinoma

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Prando, Adilson [Hospital Vera Cruz, Campinas, SP (Brazil). Dept. of Radiology and Imaging Diagnosis]. E-mail: adilson.prando@gmail.com

2008-07-15

Objective: To present computed tomography findings observed in four patients submitted to radical nephrectomy for renal cell carcinoma who developed pancreatic metastases afterwards. Materials and methods: The four patients underwent radical nephrectomy for stage Tz1 (n=2) and stage T3a (n=2) renal cell carcinoma. The mean interval between nephrectomy and detection of pancreatic metastases was eight years. Two asymptomatic patients presented with solitary pancreatic metastases (confined to the pancreas). Two symptomatic patients presented with single and multiple pancreatic metastases, both with tumor recurrence in the contralateral kidney. Results: Computed tomography studies demonstrated pancreatic metastases as solitary (n=2), single (n=1) or multiple (n=1) hypervascular lesions. Partial pancreatectomy was performed in two patients with solitary pancreatic metastases and both are free of disease at four and two years after surgery. Conclusion: Pancreatic metastases from renal cell carcinoma are rare and can occur many years after the primary tumor presentation. Multiple pancreatic metastases and pancreatic metastases associated with tumor recurrence in the contralateral kidney are uncommon. Usually, on computed tomography images pancreatic metastases are visualized as solitary hypervascular lesions, simulating isletcell tumors. Surgical management should be considered for patients with solitary pancreatic lesions. (author)

Secondary pancreatic involvement by a diffuse large B-cell lymphoma presenting as acute pancreatitis

Institute of Scientific and Technical Information of China (English)

M Wasif Saif; Sapna Khubchandani; Marek Walczak

2007-01-01

Diffuse large B-cell lymphoma is the most common type of non-Hodgkin's lymphoma. More than 50% of patients have some site of extra-nodal involvement at diagnosis,including the gastrointestinal tract and bone marrow.However, a diffuse large B-cell lymphoma presenting as acute pancreatitis is rare. A 57-year-old female presented with abdominal pain and matted lymph nodes in her axilla. She was admitted with a diagnosis of acute pancreatitis. Abdominal computed tomography (CT) scan showed diffusely enlarged pancreas due to infiltrative neoplasm and peripancreatic lymphadenopathy. Biopsy of the axillary mass revealed a large B-cell lymphoma.The patient was classified as stage Ⅳ, based on the Ann Arbor Classification, and as having a high-risk lymphoma,based on the International Prognostic Index. She was started on chemotherapy with CHOP (cyclophosphamide,doxorubicin, vincristine and prednisone). Within a week after chemotherapy, the patient's abdominal pain resolved. Follow-up CT scan of the abdomen revealed a marked decrease in the size of the pancreas and peripancreatic lymphadenopathy. A literature search revealed only seven cases of primary involvement of the pancreas in B-cell lymphoma presenting as acute pancreatitis. However, only one case of secondary pancreatic involvement by B-cell lymphoma presenting as acute pancreatitis has been published. Our case appears to be the second report of such a manifestation.Both cases responded well to chemotherapy.

Pancreatic Stellate Cells : A Starring Role in Normal and Diseased Pancreas

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Minoti eApte

2012-08-01

Full Text Available While the morphology and function of cells of the exocrine and endocrine pancreas have been studied over several centuries, one important cell type in the gland, the pancreatic stellate cell (PSC, had remained undiscovered until as recently as twenty years ago. Even after its first description in 1982, it was to be another 16 years before its biology could begin to be studied, because it was only in 1998 that methods were developed to isolate and culture PSCs from rodent and human pancreas. PSCs are now known to play a critical role in pancreatic fibrosis, a consistent histological feature of two major diseases of the pancreas - chronic pancreatitis and pancreatic cancer. In health, PSCs maintain normal tissue architecture via regulation of the synthesis and degradation of extracellular matrix (ECM proteins. Recent studies have also implied other additional functions for PSCs as progenitor cells, immune cells or intermediaries in exocrine pancreatic secretion in humans.During pancreatic injury, PSCs transform from their quiescent phase into an activated, myofibroblast-like phenotype that secretes excessive amounts of ECM proteins leading to the fibrosis of chronic pancreatitis and pancreatic cancer. An ever increasing number of factors that stimulate and/or inhibit PSC activation via paracrine and autocrine pathways are being identified and characterized. It is also now established that PSCs interact closely with pancreatic cancer cells to facilitate cancer progression. Based on these findings, several therapeutic strategies have been examined in experimental models of chronic pancreatitis as well as pancreatic cancer, in a bid to inhibit/retard PSC activation and thereby alleviate chronic pancreatitis or reduce tumour growth in pancreatic cancer. The challenge that remains is to translate these pre-clinical developments into clinically applicable treatments for patients with chronic pancreatitis and pancreatic cancer.

Acinar-to-Ductal Metaplasia Induced by Transforming Growth Factor Beta Facilitates KRASG12D-driven Pancreatic TumorigenesisSummary

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Nicolas Chuvin

2017-09-01

Full Text Available Background & Aims: Transforming growth factor beta (TGFβ acts either as a tumor suppressor or as an oncogene, depending on the cellular context and time of activation. TGFβ activates the canonical SMAD pathway through its interaction with the serine/threonine kinase type I and II heterotetrameric receptors. Previous studies investigating TGFβ-mediated signaling in the pancreas relied either on loss-of-function approaches or on ligand overexpression, and its effects on acinar cells have so far remained elusive. Methods: We developed a transgenic mouse model allowing tamoxifen-inducible and Cre-mediated conditional activation of a constitutively active type I TGFβ receptor (TβRICA in the pancreatic acinar compartment. Results: We observed that TβRICA expression induced acinar-to-ductal metaplasia (ADM reprogramming, eventually facilitating the onset of KRASG12D-induced pre-cancerous pancreatic intraepithelial neoplasia. This phenotype was characterized by the cellular activation of apoptosis and dedifferentiation, two hallmarks of ADM, whereas at the molecular level, we evidenced a modulation in the expression of transcription factors such as Hnf1β, Sox9, and Hes1. Conclusions: We demonstrate that TGFβ pathway activation plays a crucial role in pancreatic tumor initiation through its capacity to induce ADM, providing a favorable environment for KRASG12D-dependent carcinogenesis. Such findings are highly relevant for the development of early detection markers and of potentially novel treatments for pancreatic cancer patients. Keywords: Pancreas, Cancer, TGFβ, Acinar-to-Ductal Metaplasia, KRASG12D

Ginkgo Biloba Extract Kaempferol Inhibits Cell Proliferation and Induces Apoptosis in Pancreatic Cancer Cells

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Zhang, Yuqing; Chen, Aaron Y.; Li, Min; Chen, Changyi; Yao, Qizhi

2010-01-01

Background Kaempferol is one of the most important constituents in ginkgo flavonoids. Recent studies indicate kaempferol may have anti-tumor activities. The objective in this study was to determine the effect and mechanisms of kaempferol on pancreatic cancer cell proliferation and apoptosis. Materials and Methods Pancreatic cancer cell lines MIA PaCa-2 and Panc-1 were treated with Kampferol, and the inhibitory effects of kaempferol on pancreatic cancer cell proliferation were examined by direct cell counting, 3H-thymidine incorporation and MTS assay. Lactate dehydrogenase (LDH) release from cells was determined as an index of cytotoxicity. Apoptosis was analyzed by TUNEL assay. Results Upon the treatment with 70 μM kaempferol for 4 days, MIA PaCa-2 cell proliferation was significantly inhibited by 79% and 45.7% as determined by direct cell counting and MTS assay, respectively, compared with control cells (Pkaempferol significantly inhibited Panc-1 cell proliferation. Kaempferol treatment also significantly reduced 3H-thymidine incorporation in both MIA PaCa-2 and Panc-1 cells. Combination treatment of low concentrations of kaempferol and 5-fluorouracil (5-FU) showed an additive effect on the inhibition of MIA PaCa-2 cell proliferation. Furthermore, kaempferol had a significantly less cytotoxicity than 5-FU in normal human pancreatic ductal epithelial cells (P=0.029). In both MIA PaCa-2 and Panc-1 cells, apoptotic cell population was increased when treated with kaempferol in a concentration-dependent manner. Conclusions Ginkgo biloba extract kaempferol effectively inhibits pancreatic cancer cell proliferation and induces cancer cell apoptosis, which may sensitize pancreatic tumor cells to chemotherapy. Kaempferol may have clinical applications as adjuvant therapy in the treatment of pancreatic cancer. PMID:18570926

In vivo and in vitro evaluation of the effects of Urtica dioica and swimming activity on diabetic factors and pancreatic beta cells.

Science.gov (United States)

Ranjbari, Abbas; Azarbayjani, Mohammad Ali; Yusof, Ashril; Halim Mokhtar, Abdul; Akbarzadeh, Samad; Ibrahim, Mohamed Yousif; Tarverdizadeh, Bahman; Farzadinia, Parviz; Hajiaghaee, Reza; Dehghan, Firouzeh

2016-03-15

Urtica dioica (UD) has been identified as a traditional herbal medicine. This study aimed to investigate the effect of UD extract and swimming activity on diabetic parameters through in vivo and in vitro experiments. Adult WKY male rats were randomly distributed in nine groups: intact control, diabetic control, diabetic + 625 mg/kg, 1.25 g/kg UD, diabetic + 100 mg/kg Metformin, diabetic + swimming, diabetic + swimming 625 mg/kg, 1.25 g/kg UD, and diabetic +100 mg/kg Metformin + swimming. The hearts of the animals were punctured, and blood samples were collected for biochemical analysis. The entire pancreas was exposed for histologic examination. The effect of UD on insulin secretion by RIN-5F cells in 6.25 or 12.5 mM glucose dose was examined. Glucose uptake by cultured L6 myotubes was determined. The serum glucose concentration decreased, the insulin resistance and insulin sensitivity significantly increased in treated groups. These changes were more pronounced in the group that received UD extract and swimming training. Regeneration and less beta cell damage of Langerhans islets were observed in the treated groups. UD treatment increased insulin secretion in the RIN-5F cells and glucose uptake in the L6 myotubes cells. Swimming exercises accompanied by consuming UD aqueous extracts effectively improved diabetic parameters, repaired pancreatic tissues in streptozotocin-induced diabetics in vivo, and increased glucose uptake or insulin in UD-treated cells in vitro.

Pancreatic acinar cells-derived cyclophilin A promotes pancreatic damage by activating NF-κB pathway in experimental pancreatitis

International Nuclear Information System (INIS)

Yu, Ge; Wan, Rong; Hu, Yanling; Ni, Jianbo; Yin, Guojian; Xing, Miao; Shen, Jie; Tang, Maochun; Chen, Congying; Fan, Yuting; Xiao, Wenqin; Zhao, Yan; Wang, Xingpeng

2014-01-01

Highlights: • CypA is upregulated in experimental pancreatitis. • CCK induces expression and release of CypA in acinar cell in vitro. • rCypA aggravates CCK-induced acinar cell death and inflammatory cytokine production. • rCypA activates the NF-κB pathway in acinar cells in vitro. - Abstract: Inflammation triggered by necrotic acinar cells contributes to the pathophysiology of acute pancreatitis (AP), but its precise mechanism remains unclear. Recent studies have shown that Cyclophilin A (CypA) released from necrotic cells is involved in the pathogenesis of several inflammatory diseases. We therefore investigated the role of CypA in experimental AP induced by administration of sodium taurocholate (STC). CypA was markedly upregulated and widely expressed in disrupted acinar cells, infiltrated inflammatory cells, and tubular complexes. In vitro, it was released from damaged acinar cells by cholecystokinin (CCK) induction. rCypA (recombinant CypA) aggravated CCK-induced acinar cell necrosis, promoted nuclear factor (NF)-κB p65 activation, and increased cytokine production. In conclusion, CypA promotes pancreatic damage by upregulating expression of inflammatory cytokines of acinar cells via the NF-κB pathway

Pancreatic acinar cells-derived cyclophilin A promotes pancreatic damage by activating NF-κB pathway in experimental pancreatitis

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Yu, Ge [Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Wan, Rong [Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Hu, Yanling [Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Ni, Jianbo [Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Yin, Guojian; Xing, Miao [Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Shen, Jie [Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Tang, Maochun [Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Chen, Congying [Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Fan, Yuting; Xiao, Wenqin; Zhao, Yan [Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Wang, Xingpeng, E-mail: wangxingpeng@hotmail.com [Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); and others

2014-01-31

Highlights: • CypA is upregulated in experimental pancreatitis. • CCK induces expression and release of CypA in acinar cell in vitro. • rCypA aggravates CCK-induced acinar cell death and inflammatory cytokine production. • rCypA activates the NF-κB pathway in acinar cells in vitro. - Abstract: Inflammation triggered by necrotic acinar cells contributes to the pathophysiology of acute pancreatitis (AP), but its precise mechanism remains unclear. Recent studies have shown that Cyclophilin A (CypA) released from necrotic cells is involved in the pathogenesis of several inflammatory diseases. We therefore investigated the role of CypA in experimental AP induced by administration of sodium taurocholate (STC). CypA was markedly upregulated and widely expressed in disrupted acinar cells, infiltrated inflammatory cells, and tubular complexes. In vitro, it was released from damaged acinar cells by cholecystokinin (CCK) induction. rCypA (recombinant CypA) aggravated CCK-induced acinar cell necrosis, promoted nuclear factor (NF)-κB p65 activation, and increased cytokine production. In conclusion, CypA promotes pancreatic damage by upregulating expression of inflammatory cytokines of acinar cells via the NF-κB pathway.

Mitochondrial oxidative stress contributes differently to rat pancreatic islet cell apoptosis and insulin secretory defects after prolonged culture in a low non-stimulating glucose concentration.

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Roma, L P; Pascal, S M; Duprez, J; Jonas, J-C

2012-08-01

Pancreatic beta cells chronically exposed to low glucose concentrations show signs of oxidative stress, loss of glucose-stimulated insulin secretion (GSIS) and increased apoptosis. Our aim was to confirm the role of mitochondrial oxidative stress in rat islet cell apoptosis under these culture conditions and to evaluate whether its reduction similarly improves survival and GSIS. Apoptosis, oxidative stress-response gene mRNA expression and glucose-induced stimulation of mitochondrial metabolism, intracellular Ca(2+) concentration and insulin secretion were measured in male Wistar rat islets cultured for 1 week in RPMI medium containing 5-10 mmol/l glucose with or without manganese(III)tetrakis(4-benzoic acid)porphyrin (MnTBAP) or N-acetyl-L-: cysteine (NAC). Oxidative stress was measured in islet cell clusters cultured under similar conditions using cytosolic and mitochondrial redox-sensitive green fluorescent protein (roGFP1/mt-roGFP1). Prolonged culture in 5 vs 10 mmol/l glucose increased mt-roGFP1 (but not roGFP1) oxidation followed by beta cell apoptosis and loss of GSIS resulting from reduced insulin content, mitochondrial metabolism, Ca(2+) influx and Ca(2+)-induced secretion. Tolbutamide-induced, but not high K(+)-induced, Ca(2+) influx was also suppressed. Under these conditions, MnTBAP, but not NAC, triggered parallel ~50-70% reductions in mt-roGFP1 oxidation and beta cell apoptosis, but failed to protect against the loss of GSIS despite significant improvement in glucose-induced and tolbutamide-induced Ca(2+) influx. Mitochondrial oxidative stress contributes differently to rat pancreatic islet cell apoptosis and insulin secretory defects during culture in a low glucose concentration. Thus, targeting beta cell survival may not be sufficient to restore insulin secretion when beta cells suffer from prolonged mitochondrial oxidative stress, e.g. in the context of reduced glucose metabolism.

α-Lipoic acid prevents lipotoxic cardiomyopathy in acyl CoA-synthase transgenic mice

International Nuclear Information System (INIS)

Lee, Young; Naseem, R. Haris; Park, Byung-Hyun; Garry, Daniel J.; Richardson, James A.; Schaffer, Jean E.; Unger, Roger H.

2006-01-01

α-Lipoic acid (α-LA) mimics the hypothalamic actions of leptin on food intake, energy expenditure, and activation of AMP-activated protein kinase (AMPK). To determine if, like leptin, α-LA protects against cardiac lipotoxicity, α-LA was fed to transgenic mice with cardiomyocyte-specific overexpression of the acyl CoA synthase (ACS) gene. Untreated ACS-transgenic mice died prematurely with increased triacylglycerol content and dilated cardiomyopathy, impaired systolic function and myofiber disorganization, apoptosis, and interstitial fibrosis on microscopy. In α-LA-treated ACS-transgenic mice heart size, echocardiogram and TG content were normal. Plasma TG fell 50%, hepatic-activated phospho-AMPK rose 6-fold, sterol regulatory element-binding protein-1c declined 50%, and peroxisome proliferator-activated receptor-γ cofactor-1α mRNA rose 4-fold. Since food restriction did not prevent lipotoxicity, we conclude that α-LA treatment, like hyperleptinemia, protects the heart of ACS-transgenic mice from lipotoxicity

Serum levels of pancreatic stone protein (PSP/reg1A as an indicator of beta-cell apoptosis suggest an increased apoptosis rate in hepatocyte nuclear factor 1 alpha (HNF1A-MODY carriers from the third decade of life onward

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Bacon Siobhan

2012-07-01

Full Text Available Abstract Background Mutations in the transcription factor hepatocyte nuclear factor-1-alpha (HNF1A result in the commonest type of maturity onset diabetes of the young (MODY. HNF1A-MODY carriers have reduced pancreatic beta cell mass, partially due to an increased rate of apoptosis. To date, it has not been possible to determine when apoptosis is occurring in HNF1A-MODY.We have recently demonstrated that beta cell apoptosis stimulates the expression of the pancreatic stone protein/regenerating (PSP/reg gene in surviving neighbour cells, and that PSP/reg1A protein is subsequently secreted from these cells. The objective of this study was to determine whether serum levels of PSP/reg1A are elevated during disease progression in HNF1A-MODY carriers, and whether it may provide information regarding the onset of beta-cell apoptosis. Methods We analysed serum PSP/reg1A levels and correlated with clinical and biochemical parameters in subjects with HNF1A-MODY, glucokinase (GCK-MODY, and type 1 diabetes mellitus. A control group of normoglycaemic subjects was also analysed. Results PSP/reg1A serum levels were significantly elevated in HNF1A-MODY (n = 37 subjects compared to controls (n = 60 (median = 12.50 ng/ml, IQR = 10.61-17.87 ng/ml versus median = 10.72 ng/ml, IQR = 8.94-12.54 ng/ml, p = 0.0008. PSP/reg1A correlated negatively with insulin levels during OGTT, (rho = −0.40, p = 0.02. Interestingly we noted a significant positive correlation of PSP/reg1A with age of the HNF1A-MODY carriers (rho = 0.40 p = 0.02 with an age of 25 years separating carriers with low and high PSP/reg1A levels. Patients with type 1 diabetes mellitus also had elevated serum levels of PSP/reg1A compared to controls, however this was independent of the duration of diabetes. Conclusion Our data suggest that beta cell apoptosis contributes increasingly to the pathophysiology of HNF1A-MODY in patients 25 years and over

Serum levels of pancreatic stone protein (PSP)/reg1A as an indicator of beta-cell apoptosis suggest an increased apoptosis rate in hepatocyte nuclear factor 1 alpha (HNF1A-MODY) carriers from the third decade of life onward

LENUS (Irish Health Repository)

Bacon, Siobhan

2012-07-18

AbstractBackgroundMutations in the transcription factor hepatocyte nuclear factor-1-alpha (HNF1A) result in the commonest type of maturity onset diabetes of the young (MODY). HNF1A-MODY carriers have reduced pancreatic beta cell mass, partially due to an increased rate of apoptosis. To date, it has not been possible to determine when apoptosis is occurring in HNF1A-MODY.We have recently demonstrated that beta cell apoptosis stimulates the expression of the pancreatic stone protein\\/regenerating (PSP\\/reg) gene in surviving neighbour cells, and that PSP\\/reg1A protein is subsequently secreted from these cells. The objective of this study was to determine whether serum levels of PSP\\/reg1A are elevated during disease progression in HNF1A-MODY carriers, and whether it may provide information regarding the onset of beta-cell apoptosis.MethodsWe analysed serum PSP\\/reg1A levels and correlated with clinical and biochemical parameters in subjects with HNF1A-MODY, glucokinase (GCK-MODY), and type 1 diabetes mellitus. A control group of normoglycaemic subjects was also analysed.ResultsPSP\\/reg1A serum levels were significantly elevated in HNF1A-MODY (n = 37) subjects compared to controls (n = 60) (median = 12.50 ng\\/ml, IQR = 10.61-17.87 ng\\/ml versus median = 10.72 ng\\/ml, IQR = 8.94-12.54 ng\\/ml, p = 0.0008). PSP\\/reg1A correlated negatively with insulin levels during OGTT, (rho = −0.40, p = 0.02). Interestingly we noted a significant positive correlation of PSP\\/reg1A with age of the HNF1A-MODY carriers (rho = 0.40 p = 0.02) with an age of 25 years separating carriers with low and high PSP\\/reg1A levels. Patients with type 1 diabetes mellitus also had elevated serum levels of PSP\\/reg1A compared to controls, however this was independent of the duration of diabetes.ConclusionOur data suggest that beta cell apoptosis contributes increasingly to the pathophysiology of HNF1A-MODY in patients 25 years and

Combined lipidomic and proteomic analysis of isolated human islets exposed to palmitate reveals time-dependent changes in insulin secretion and lipid metabolism.

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Kirsten Roomp

Full Text Available Studies on the pathophysiology of type 2 diabetes mellitus (T2DM have linked the accumulation of lipid metabolites to the development of beta-cell dysfunction and impaired insulin secretion. In most in vitro models of T2DM, rodent islets or beta-cell lines are used and typically focus is on specific cellular pathways or organs. Our aim was to, firstly, develop a combined lipidomics and proteomics approach for lipotoxicity in isolated human islets and, secondly, investigate if the approach could delineate novel and/ or confirm reported mechanisms of lipotoxicity. To this end isolated human pancreatic islets, exposed to chronically elevated palmitate concentrations for 0, 2 and 7 days, were functionally characterized and their levels of multiple targeted lipid and untargeted protein species determined. Glucose-stimulated insulin secretion from the islets increased on day 2 and decreased on day 7. At day 7 islet insulin content decreased and the proinsulin to insulin content ratio doubled. Amounts of cholesterol, stearic acid, C16 dihydroceramide and C24:1 sphingomyelin, obtained from the lipidomic screen, increased time-dependently in the palmitate-exposed islets. The proteomic screen identified matching changes in proteins involved in lipid biosynthesis indicating up-regulated cholesterol and lipid biosynthesis in the islets. Furthermore, proteins associated with immature secretory granules were decreased when palmitate exposure time was increased despite their high affinity for cholesterol. Proteins associated with mature secretory granules remained unchanged. Pathway analysis based on the protein and lipid expression profiles implicated autocrine effects of insulin in lipotoxicity. Taken together the study demonstrates that combining different omics approaches has potential in mapping of multiple simultaneous cellular events. However, it also shows that challenges exist for effectively combining lipidomics and proteomics in primary cells. Our

Interaction of Stellate Cells with Pancreatic Carcinoma Cells

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Marco Siech

2010-09-01

Full Text Available Pancreatic cancer is characterized by its late detection, aggressive growth, intense infiltration into adjacent tissue, early metastasis, resistance to chemo- and radiotherapy and a strong “desmoplastic reaction”. The dense stroma surrounding carcinoma cells is composed of fibroblasts, activated stellate cells (myofibroblast-like cells, various inflammatory cells, proliferating vascular structures, collagens and fibronectin. In particular the cellular components of the stroma produce the tumor microenvironment, which plays a critical role in tumor growth, invasion, spreading, metastasis, angiogenesis, inhibition of anoikis, and chemoresistance. Fibroblasts, myofibroblasts and activated stellate cells produce the extracellular matrix components and are thought to interact actively with tumor cells, thereby promoting cancer progression. In this review, we discuss our current understanding of the role of pancreatic stellate cells (PSC in the desmoplastic response of pancreas cancer and the effects of PSC on tumor progression, metastasis and drug resistance. Finally we present some novel ideas for tumor therapy by interfering with the cancer cell-host interaction.

Exosomes derived from pancreatic cancer cells induce activation and profibrogenic activities in pancreatic stellate cells.

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Masamune, Atsushi; Yoshida, Naoki; Hamada, Shin; Takikawa, Tetsuya; Nabeshima, Tatsuhide; Shimosegawa, Tooru

2018-01-01

Pancreatic cancer cells (PCCs) interact with pancreatic stellate cells (PSCs), which play a pivotal role in pancreatic fibrogenesis, to develop the cancer-conditioned tumor microenvironment. Exosomes are membrane-enclosed nanovesicles, and have been increasingly recognized as important mediators of cell-to-cell communications. The aim of this study was to clarify the effects of PCC-derived exosomes on cell functions in PSCs. Exosomes were isolated from the conditioned medium of Panc-1 and SUIT-2 PCCs. Human primary PSCs were treated with PCC-derived exosomes. PCC-derived exosomes stimulated the proliferation, migration, activation of ERK and Akt, the mRNA expression of α-smooth muscle actin (ACTA2) and fibrosis-related genes, and procollagen type I C-peptide production in PSCs. Ingenuity pathway analysis of the microarray data identified transforming growth factor β1 and tumor necrosis factor as top upstream regulators. PCCs increased the expression of miR-1246 and miR-1290, abundantly contained in PCC-derived exosomes, in PSCs. Overexpression of miR-1290 induced the expression of ACTA2 and fibrosis-related genes in PSCs. In conclusion, PCC-derived exosomes stimulate activation and profibrogenic activities in PSCs. Exosome-mediated interactions between PSCs and PCCs might play a role in the development of the tumor microenvironment. Copyright © 2017 Elsevier Inc. All rights reserved.

Role of the mitochondria in immune-mediated apoptotic death of the human pancreatic β cell line βLox5.

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Yaíma L Lightfoot

Full Text Available Mitochondria are indispensable in the life and death of many types of eukaryotic cells. In pancreatic beta cells, mitochondria play an essential role in the secretion of insulin, a hormone that regulates blood glucose levels. Unregulated blood glucose is a hallmark symptom of diabetes. The onset of Type 1 diabetes is preceded by autoimmune-mediated destruction of beta cells. However, the exact role of mitochondria has not been assessed in beta cell death. In this study, we examine the role of mitochondria in both Fas- and proinflammatory cytokine-mediated destruction of the human beta cell line, βLox5. IFNγ primed βLox5 cells for apoptosis by elevating cell surface Fas. Consequently, βLox5 cells were killed by caspase-dependent apoptosis by agonistic activation of Fas, but only after priming with IFNγ. This beta cell line undergoes both apoptotic and necrotic cell death after incubation with the combination of the proinflammatory cytokines IFNγ and TNFα. Additionally, both caspase-dependent and -independent mechanisms that require proper mitochondrial function are involved. Mitochondrial contributions to βLox5 cell death were analyzed using mitochondrial DNA (mtDNA depleted βLox5 cells, or βLox5 ρ(0 cells. βLox5 ρ(0 cells are not sensitive to IFNγ and TNFα killing, indicating a direct role for the mitochondria in cytokine-induced cell death of the parental cell line. However, βLox5 ρ(0 cells are susceptible to Fas killing, implicating caspase-dependent extrinsic apoptotic death is the mechanism by which these human beta cells die after Fas ligation. These data support the hypothesis that immune mediators kill βLox5 cells by both mitochondrial-dependent intrinsic and caspase-dependent extrinsic pathways.

Endothelial Cells Control Pancreatic Cell Fate at Defined Stages through EGFL7 Signaling

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Der-I Kao

2015-02-01

Full Text Available Although endothelial cells have been shown to affect mouse pancreatic development, their precise function in human development remains unclear. Using a coculture system containing human embryonic stem cell (hESC-derived progenitors and endothelial cells, we found that endothelial cells play a stage-dependent role in pancreatic development, in which they maintain pancreatic progenitor (PP self-renewal and impair further differentiation into hormone-expressing cells. The mechanistic studies suggest that the endothelial cells act through the secretion of EGFL7. Consistently, endothelial overexpression of EGFL7 in vivo using a transgenic mouse model resulted in an increase of PP proliferation rate and a decrease of differentiation toward endocrine cells. These studies not only identified the role of EGFL7 as the molecular handle involved in the crosstalk between endothelium and pancreatic epithelium, but also provide a paradigm for using hESC stepwise differentiation to dissect the stage-dependent roles of signals controlling organogenesis.

Timing of Ca2+ response in pancreatic beta-cells is related to mitochondrial mass

DEFF Research Database (Denmark)

Gustavsson, N; Abedi, G; Larsson-Nyrén, G

2006-01-01

timing are disturbed in beta-cells from hyperglycemic mice and one of the causes is likely to be an altered mitochondrial metabolism. Mitochondria play a key role in the control of nutrient-induced insulin secretion. Here, we used confocal microscopy with the fluorescent probe MitoTracker Red CMXRos...

AMP-activated protein kinase (AMPK mediates nutrient regulation of thioredoxin-interacting protein (TXNIP in pancreatic beta-cells.

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Maayan Shaked

Full Text Available Thioredoxin-interacting protein (TXNIP regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. We found that both palmitate and oleate inhibited TXNIP in a rat beta-cell line and islets. Palmitate inhibition of TXNIP was independent of fatty acid beta-oxidation or esterification. AMP-activated protein kinase (AMPK has an important role in cellular energy sensing and control of metabolic homeostasis; therefore we investigated its involvement in nutrient regulation of TXNIP. As expected, glucose inhibited whereas palmitate stimulated AMPK. Pharmacologic activators of AMPK mimicked fatty acids by inhibiting TXNIP. AMPK knockdown increased TXNIP expression in presence of high glucose with and without palmitate, indicating that nutrient (glucose and fatty acids effects on TXNIP are mediated in part via modulation of AMPK activity. TXNIP is transcriptionally regulated by carbohydrate response element-binding protein (ChREBP. Palmitate inhibited glucose-stimulated ChREBP nuclear entry and recruitment to the Txnip promoter, thereby inhibiting Txnip transcription. We conclude that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment

Chaperones ameliorate beta cell dysfunction associated with human islet amyloid polypeptide overexpression.

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Lisa Cadavez

Full Text Available In type 2 diabetes, beta-cell dysfunction is thought to be due to several causes, one being the formation of toxic protein aggregates called islet amyloid, formed by accumulations of misfolded human islet amyloid polypeptide (hIAPP. The process of hIAPP misfolding and aggregation is one of the factors that may activate the unfolded protein response (UPR, perturbing endoplasmic reticulum (ER homeostasis. Molecular chaperones have been described to be important in regulating ER response to ER stress. In the present work, we evaluate the role of chaperones in a stressed cellular model of hIAPP overexpression. A rat pancreatic beta-cell line expressing hIAPP exposed to thapsigargin or treated with high glucose and palmitic acid, both of which are known ER stress inducers, showed an increase in ER stress genes when compared to INS1E cells expressing rat IAPP or INS1E control cells. Treatment with molecular chaperone glucose-regulated protein 78 kDa (GRP78, also known as BiP or protein disulfite isomerase (PDI, and chemical chaperones taurine-conjugated ursodeoxycholic acid (TUDCA or 4-phenylbutyrate (PBA, alleviated ER stress and increased insulin secretion in hIAPP-expressing cells. Our results suggest that the overexpression of hIAPP induces a stronger response of ER stress markers. Moreover, endogenous and chemical chaperones are able to ameliorate induced ER stress and increase insulin secretion, suggesting that improving chaperone capacity can play an important role in improving beta-cell function in type 2 diabetes.

The expression of dominant negative TCF7L2 in pancreatic beta cells during the embryonic stage causes impaired glucose homeostasis

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Weijuan Shao

2015-04-01

Conclusions: Our observations support a cell autonomous role for TCF7L2 in pancreatic β-cells suggested by most, though not all, investigations. βTCFDN is a novel model for further exploring the role of TCF7L2 in β-cell genesis and metabolic homeostasis.

Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models

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Wen, Li; Voronina, Svetlana; Javed, Muhammad A.; Awais, Muhammad; Szatmary, Peter; Latawiec, Diane; Chvanov, Michael; Collier, David; Huang, Wei; Barrett, John; Begg, Malcolm; Stauderman, Ken; Roos, Jack; Grigoryev, Sergey; Ramos, Stephanie; Rogers, Evan; Whitten, Jeff; Velicelebi, Gonul; Dunn, Michael; Tepikin, Alexei V.; Criddle, David N.; Sutton, Robert

2015-01-01

Background & Aims Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release–activated calcium modulator ORAI1 is the most abundant Ca2+ entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice. Methods Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects. Results GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca2+ currents after Ca2+ release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis. Conclusions Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed

SEC23B is required for pancreatic acinar cell function in adult mice

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Khoriaty, Rami; Vogel, Nancy; Hoenerhoff, Mark J.; Sans, M. Dolors; Zhu, Guojing; Everett, Lesley; Nelson, Bradley; Durairaj, Haritha; McKnight, Brooke; Zhang, Bin; Ernst, Stephen A.; Ginsburg, David; Williams, John A.

2017-01-01

Mice with germline absence of SEC23B die perinatally, exhibiting massive pancreatic degeneration. We generated mice with tamoxifen-inducible, pancreatic acinar cell–specific Sec23b deletion. Inactivation of Sec23b exclusively in the pancreatic acinar cells of adult mice results in decreased overall pancreatic weights from pancreatic cell loss (decreased pancreatic DNA, RNA, and total protein content), as well as degeneration of exocrine cells, decreased zymogen granules, and alterations in the endoplasmic reticulum (ER), ranging from vesicular ER to markedly expanded cisternae with accumulation of moderate-density content or intracisternal granules. Acinar Sec23b deletion results in induction of ER stress and increased apoptosis in the pancreas, potentially explaining the loss of pancreatic cells and decreased pancreatic weight. These findings demonstrate that SEC23B is required for normal function of pancreatic acinar cells in adult mice. PMID:28539403

Histone deacetylases 1 and 3 but not 2 mediate cytokine-induced beta cell apoptosis in INS-1 cells and dispersed primary islets from rats and are differentially regulated in the islets of type 1 diabetic children

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Lundh, M; Christensen, D P; Damgaard Nielsen, M

2012-01-01

onset, HDAC1 was upregulated in beta cells whereas HDAC2 and -3 were downregulated in comparison with five paediatric controls. CONCLUSIONS/INTERPRETATION: These data demonstrate non-redundant functions of islet class I HDACs and suggest that targeting HDAC1 and HDAC3 would provide optimal protection......AIMS/HYPOTHESIS: Histone deacetylases (HDACs) are promising pharmacological targets in cancer and autoimmune diseases. All 11 classical HDACs (HDAC1-11) are found in the pancreatic beta cell, and HDAC inhibitors (HDACi) protect beta cells from inflammatory insults. We investigated which HDACs...... of HDAC1, -2 and -3 rescued INS-1 cells from inflammatory damage. Small hairpin RNAs against HDAC1 and -3, but not HDAC2, reduced pro-inflammatory cytokine-induced beta cell apoptosis in INS-1 and primary rat islets. The protective properties of specific HDAC knock-down correlated with attenuated cytokine...

Adipose Stem Cell Therapy Mitigates Chronic Pancreatitis via Differentiation into Acinar-like Cells in Mice.

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Sun, Zhen; Gou, Wenyu; Kim, Do-Sung; Dong, Xiao; Strange, Charlie; Tan, Yu; Adams, David B; Wang, Hongjun

2017-11-01

The objective of this study was to assess the capacity of adipose-derived mesenchymal stem cells (ASCs) to mitigate disease progression in an experimental chronic pancreatitis mouse model. Chronic pancreatitis (CP) was induced in C57BL/6 mice by repeated ethanol and cerulein injection, and mice were then infused with 4 × 10 5 or 1 × 10 6 GFP + ASCs. Pancreas morphology, fibrosis, inflammation, and presence of GFP + ASCs in pancreases were assessed 2 weeks after treatment. We found that ASC infusion attenuated pancreatic damage, preserved pancreas morphology, and reduced pancreatic fibrosis and cell death. GFP + ASCs migrated to pancreas and differentiated into amylase + cells. In further confirmation of the plasticity of ASCs, ASCs co-cultured with acinar cells in a Transwell system differentiated into amylase + cells with increased expression of acinar cell-specific genes including amylase and chymoB1. Furthermore, culture of acinar or pancreatic stellate cell lines in ASC-conditioned medium attenuated ethanol and cerulein-induced pro-inflammatory cytokine production in vitro. Our data show that a single intravenous injection of ASCs ameliorated CP progression, likely by directly differentiating into acinar-like cells and by suppressing inflammation, fibrosis, and pancreatic tissue damage. These results suggest that ASC cell therapy has the potential to be a valuable treatment for patients with pancreatitis. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Beta cell 5'-shifted isomiRs are candidate regulatory hubs in type 2 diabetes.

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Jeanette Baran-Gale

Full Text Available Next-generation deep sequencing of small RNAs has unveiled the complexity of the microRNA (miRNA transcriptome, which is in large part due to the diversity of miRNA sequence variants ("isomiRs". Changes to a miRNA's seed sequence (nucleotides 2-8, including shifted start positions, can redirect targeting to a dramatically different set of RNAs and alter biological function. We performed deep sequencing of small RNA from mouse insulinoma (MIN6 cells (widely used as a surrogate for the study of pancreatic beta cells and developed a bioinformatic analysis pipeline to profile isomiR diversity. Additionally, we applied the pipeline to recently published small RNA-seq data from primary human beta cells and whole islets and compared the miRNA profiles with that of MIN6. We found that: (1 the miRNA expression profile in MIN6 cells is highly correlated with those of primary human beta cells and whole islets; (2 miRNA loci can generate multiple highly expressed isomiRs with different 5'-start positions (5'-isomiRs; (3 isomiRs with shifted start positions (5'-shifted isomiRs are highly expressed, and can be as abundant as their unshifted counterparts (5'-reference miRNAs. Finally, we identified 10 beta cell miRNA families as candidate regulatory hubs in a type 2 diabetes (T2D gene network. The most significant candidate hub was miR-29, which we demonstrated regulates the mRNA levels of several genes critical to beta cell function and implicated in T2D. Three of the candidate miRNA hubs were novel 5'-shifted isomiRs: miR-375+1, miR-375-1 and miR-183-5p+1. We showed by in silico target prediction and in vitro transfection studies that both miR-375+1 and miR-375-1 are likely to target an overlapping, but distinct suite of beta cell genes compared to canonical miR-375. In summary, this study characterizes the isomiR profile in beta cells for the first time, and also highlights the potential functional relevance of 5'-shifted isomiRs to T2D.

Studies of insulin secretory responses and of arachidonic acid incorporation into phospholipids of stably transfected insulinoma cells that overexpress group VIA phospholipase A2 (iPLA2beta ) indicate a signaling rather than a housekeeping role for iPLA2beta.

Science.gov (United States)

Ma, Z; Ramanadham, S; Wohltmann, M; Bohrer, A; Hsu, F F; Turk, J

2001-04-20

A cytosolic 84-kDa group VIA phospholipase A(2) (iPLA(2)beta) that does not require Ca(2+) for catalysis has been cloned from several sources, including rat and human pancreatic islet beta-cells and murine P388D1 cells. Many potential iPLA(2)beta functions have been proposed, including a signaling role in beta-cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA(2)beta function rest in part on effects of inhibiting iPLA(2)beta activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A(2). Manipulation of iPLA(2)beta expression by molecular biologic means is an alternative approach to study iPLA(2)beta functions, and we have used a retroviral construct containing iPLA(2)beta cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA(2)beta. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA(2)beta-overexpressing lines exhibit amplified insulin secretory responses to glucose and cAMP-elevating agents, and BEL substantially attenuates stimulated secretion. Electrospray ionization mass spectrometric analyses of arachidonic acid incorporation into INS-1 cell PC indicate that neither overexpression nor inhibition of iPLA(2)beta affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA(2)beta indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA(2)beta associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA(2)beta in insulin-secreting beta-cells.

In vitro proliferation of adult human beta-cells.

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Sabine Rutti

Full Text Available A decrease in functional beta-cell mass is a key feature of type 2 diabetes. Glucagon-like peptide 1 (GLP-1 analogues induce proliferation of rodent beta-cells. However, the proliferative capacity of human beta-cells and its modulation by GLP-1 analogues remain to be fully investigated. We therefore sought to quantify adult human beta-cell proliferation in vitro and whether this is affected by the GLP-1 analogue liraglutide.Human islets from 7 adult cadaveric organ donors were dispersed into single cells. Beta-cells were purified by FACS. Non-sorted cells and the beta-cell enriched ("beta-cells" population were plated on extracellular matrix from rat (804G and human bladder carcinoma cells (HTB9 or bovine corneal endothelial ECM (BCEC. Cells were maintained in culture+/-liraglutide for 4 days in the presence of BrdU.Rare human beta-cell proliferation could be observed either in the purified beta-cell population (0.051±0.020%; 22 beta-cells proliferating out of 84'283 beta-cells counted or in the non-sorted cell population (0.055±0.011%; 104 proliferating beta-cells out of 232'826 beta-cells counted, independently of the matrix or the culture conditions. Liraglutide increased human beta-cell proliferation on BCEC in the non-sorted cell population (0.082±0.034% proliferating beta-cells vs. 0.017±0.008% in control, p<0.05.These results indicate that adult human beta-cell proliferation can occur in vitro but remains an extremely rare event with these donors and particular culture conditions. Liraglutide increases beta-cell proliferation only in the non-sorted cell population and only on BCEC. However, it cannot be excluded that human beta-cells may proliferate to a greater extent in situ in response to natural stimuli.

Lysosome-Associated Membrane Proteins (LAMP Maintain Pancreatic Acinar Cell Homeostasis: LAMP-2–Deficient Mice Develop PancreatitisSummary

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Olga A. Mareninova

2015-11-01

Full Text Available Background & Aims: The pathogenic mechanism of pancreatitis is poorly understood. Recent evidence implicates defective autophagy in pancreatitis responses; however, the pathways mediating impaired autophagy in pancreas remain largely unknown. Here, we investigate the role of lysosome associated membrane proteins (LAMPs in pancreatitis. Methods: We analyzed changes in LAMPs in experimental models and human pancreatitis, and the underlying mechanisms: LAMP deglycosylation and degradation. LAMP cleavage by cathepsin B (CatB was analyzed by mass spectrometry. We used mice deficient in LAMP-2 to assess its role in pancreatitis. Results: Pancreatic levels of LAMP-1 and LAMP-2 greatly decrease across various pancreatitis models and in human disease. Pancreatitis does not trigger the LAMPs’ bulk deglycosylation but induces their degradation via CatB-mediated cleavage of the LAMP molecule close to the boundary between luminal and transmembrane domains. LAMP-2 null mice spontaneously develop pancreatitis that begins with acinar cell vacuolization due to impaired autophagic flux, and progresses to severe pancreas damage characterized by trypsinogen activation, macrophage-driven inflammation, and acinar cell death. LAMP-2 deficiency causes a decrease in pancreatic digestive enzymes content, and stimulates the basal and inhibits cholecystokinin-induced amylase secretion by acinar cells. The effects of LAMP-2 knockout and acute cerulein pancreatitis overlap, which corroborates the pathogenic role of LAMP decrease in experimental pancreatitis models. Conclusions: The results indicate a critical role for LAMPs, particularly LAMP-2, in maintaining pancreatic acinar cell homeostasis and provide evidence that defective lysosomal function, resulting in impaired autophagy, leads to pancreatitis. Mice with LAMP-2 deficiency present a novel genetic model of human pancreatitis caused by lysosomal/autophagic dysfunction. Keywords: Amylase Secretion, Autophagy

Imaging of gene expression in live pancreatic islet cell lines using dual-isotope SPECT.

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Tai, Joo Ho; Nguyen, Binh; Wells, R Glenn; Kovacs, Michael S; McGirr, Rebecca; Prato, Frank S; Morgan, Timothy G; Dhanvantari, Savita

2008-01-01

We are combining nuclear medicine with molecular biology to establish a sensitive, quantitative, and tomographic method with which to detect gene expression in pancreatic islet cells in vivo. Dual-isotope SPECT can be used to image multiple molecular events simultaneously, and coregistration of SPECT and CT images enables visualization of reporter gene expression in the correct anatomic context. We have engineered pancreatic islet cell lines for imaging with SPECT/CT after transplantation under the kidney capsule. INS-1 832/13 and alphaTC1-6 cells were stably transfected with a herpes simplex virus type 1-thymidine kinase-green fluorescent protein (HSV1-thymidine kinase-GFP) fusion construct (tkgfp). After clonal selection, radiolabel uptake was determined by incubation with 5-(131)I-iodo-1-(2-deoxy-2-fluoro-beta-d-arabinofuranosyl)uracil ((131)I-FIAU) (alphaTC1-6 cells) or (123)I-FIAU (INS-1 832/13 cells). For the first set of in vivo experiments, SPECT was conducted after alphaTC1-6/tkgfp cells had been labeled with either (131)I-FIAU or (111)In-tropolone and transplanted under the left kidney capsule of CD1 mice. Reconstructed SPECT images were coregistered to CT. In a second study using simultaneous acquisition dual-isotope SPECT, INS-1 832/13 clone 9 cells were labeled with (111)In-tropolone before transplantation. Mice were then systemically administered (123)I-FIAU and data for both (131)I and (111)In were acquired simultaneously. alphaTC1-6/tkgfp cells showed a 15-fold greater uptake of (131)I-FIAU, and INS-1/tkgfp cells showed a 12-fold greater uptake of (123)I-FIAU, compared with that of wild-type cells. After transplantation under the kidney capsule, both reporter gene expression and location of cells could be visualized in vivo with dual-isotope SPECT. Immunohistochemistry confirmed the presence of glucagon- and insulin-positive cells at the site of transplantation. Dual-isotope SPECT is a promising method to detect gene expression in and location of

Expression of human cationic trypsinogen (PRSS1) in murine acinar cells promotes pancreatitis and apoptotic cell death

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Athwal, T; Huang, W; Mukherjee, R; Latawiec, D; Chvanov, M; Clarke, R; Smith, K; Campbell, F; Merriman, C; Criddle, D; Sutton, R; Neoptolemos, J; Vlatković, N

2014-01-01

Hereditary pancreatitis (HP) is an autosomal dominant disease that displays the features of both acute and chronic pancreatitis. Mutations in human cationic trypsinogen (PRSS1) are associated with HP and have provided some insight into the pathogenesis of pancreatitis, but mechanisms responsible for the initiation of pancreatitis have not been elucidated and the role of apoptosis and necrosis has been much debated. However, it has been generally accepted that trypsinogen, prematurely activated within the pancreatic acinar cell, has a major role in the initiation process. Functional studies of HP have been limited by the absence of an experimental system that authentically mimics disease development. We therefore developed a novel transgenic murine model system using wild-type (WT) human PRSS1 or two HP-associated mutants (R122H and N29I) to determine whether expression of human cationic trypsinogen in murine acinar cells promotes pancreatitis. The rat elastase promoter was used to target transgene expression to pancreatic acinar cells in three transgenic strains that were generated: Tg(Ela-PRSS1)NV, Tg(Ela-PRSS1*R122H)NV and Tg(Ela-PRSS1*N29I)NV. Mice were analysed histologically, immunohistochemically and biochemically. We found that transgene expression is restricted to pancreatic acinar cells and transgenic PRSS1 proteins are targeted to the pancreatic secretory pathway. Animals from all transgenic strains developed pancreatitis characterised by acinar cell vacuolisation, inflammatory infiltrates and fibrosis. Transgenic animals also developed more severe pancreatitis upon treatment with low-dose cerulein than controls, displaying significantly higher scores for oedema, inflammation and overall histopathology. Expression of PRSS1, WT or mutant, in acinar cells increased apoptosis in pancreatic tissues and isolated acinar cells. Moreover, studies of isolated acinar cells demonstrated that transgene expression promotes apoptosis rather than necrosis. We therefore

Inhibition of Cell Survival by Curcumin Is Associated with Downregulation of Cell Division Cycle 20 (Cdc20) in Pancreatic Cancer Cells.

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Zhang, Yu; Xue, Ying-Bo; Li, Hang; Qiu, Dong; Wang, Zhi-Wei; Tan, Shi-Sheng

2017-02-04

Pancreatic cancer is one of the most aggressive human tumors in the United States. Curcumin, a polyphenol derived from the Curcuma longa plant, has been reported to exert its antitumor activity in pancreatic cancer. However, the molecular mechanisms of curcumin-mediated tumor suppressive function have not been fully elucidated. In the current study, we explore whether curcumin exhibits its anti-cancer function through inhibition of oncoprotein cell division cycle 20 (Cdc20) in pancreatic cancer cells. We found that curcumin inhibited cell growth, enhanced apoptosis, induced cell cycle arrest and retarded cell invasion in pancreatic cancer cells. Moreover, we observed that curcumin significantly inhibited the expression of Cdc20 in pancreatic cancer cells. Furthermore, our results demonstrated that overexpression of Cdc20 enhanced cell proliferation and invasion, and abrogated the cytotoxic effects induced by curcumin in pancreatic cancer cells. Consistently, downregulation of Cdc20 promoted curcumin-mediated anti-tumor activity. Therefore, our findings indicated that inhibition of Cdc20 by curcumin could be useful for the treatment of pancreatic cancer patients.

Using CRISPR/Cas9 to Knock out Amylase in Acinar Cells Decreases Pancreatitis-Induced Autophagy

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Kohei Yasunaga

2018-01-01

Full Text Available Pancreatic cancer is a malignant neoplasm that originates from acinar cells. Acinar cells get reprogrammed to become duct cells, resulting in pancreatic cancer. Pancreatitis is an acinar cell inflammation, leading to “impaired autophagy flux”. Pancreatitis promotes acinar-to-ductal transdifferentiation. Expression of amylase gets eliminated during the progression of pancreatic cancer. Amylase is considered as an acinar cell marker; however, its function in cells is not known. Thus, we investigated whether amylase affects the acinar cell autophagy and whether it plays any role in development of pancreatitis. Here, we knocked out ATG12 in a pancreatic cancer cells and acinar cells using CRISPR/Cas9. Autophagy inhibition led to an increase in the expression of duct cell markers and a simultaneous decrease in that of acinar cell markers. It also caused an increase in cell viability and changes in mitochondrial morphology. Next, we knocked out amylase in acinar cells. Amylase deficiency decreased autophagy induced by pancreatitis. Our results suggest that amylase controls pancreatitis-induced autophagy. We found that eliminating amylase expression contributes to pancreatic cancer etiology by decreasing autophagy. Furthermore, our results indicate that amylase plays a role in selective pancreatitis-induced autophagy of pancreatic enzyme vesicles.

Gene expression patterns in pancreatic tumors, cells and tissues.

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Anson W Lowe

2007-03-01

Full Text Available Cancers of the pancreas originate from both the endocrine and exocrine elements of the organ, and represent a major cause of cancer-related death. This study provides a comprehensive assessment of gene expression for pancreatic tumors, the normal pancreas, and nonneoplastic pancreatic disease.DNA microarrays were used to assess the gene expression for surgically derived pancreatic adenocarcinomas, islet cell tumors, and mesenchymal tumors. The addition of normal pancreata, isolated islets, isolated pancreatic ducts, and pancreatic adenocarcinoma cell lines enhanced subsequent analysis by increasing the diversity in gene expression profiles obtained. Exocrine, endocrine, and mesenchymal tumors displayed unique gene expression profiles. Similarities in gene expression support the pancreatic duct as the origin of adenocarcinomas. In addition, genes highly expressed in other cancers and associated with specific signal transduction pathways were also found in pancreatic tumors.The scope of the present work was enhanced by the inclusion of publicly available datasets that encompass a wide spectrum of human tissues and enabled the identification of candidate genes that may serve diagnostic and therapeutic goals.

TLR9 ligation in pancreatic stellate cells promotes tumorigenesis.

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Zambirinis, Constantinos P; Levie, Elliot; Nguy, Susanna; Avanzi, Antonina; Barilla, Rocky; Xu, Yijie; Seifert, Lena; Daley, Donnele; Greco, Stephanie H; Deutsch, Michael; Jonnadula, Saikiran; Torres-Hernandez, Alejandro; Tippens, Daniel; Pushalkar, Smruti; Eisenthal, Andrew; Saxena, Deepak; Ahn, Jiyoung; Hajdu, Cristina; Engle, Dannielle D; Tuveson, David; Miller, George

2015-11-16

Modulation of Toll-like receptor (TLR) signaling can have protective or protumorigenic effects on oncogenesis depending on the cancer subtype and on specific inflammatory elements within the tumor milieu. We found that TLR9 is widely expressed early during the course of pancreatic transformation and that TLR9 ligands are ubiquitous within the tumor microenvironment. TLR9 ligation markedly accelerates oncogenesis, whereas TLR9 deletion is protective. We show that TLR9 activation has distinct effects on the epithelial, inflammatory, and fibrogenic cellular subsets in pancreatic carcinoma and plays a central role in cross talk between these compartments. Specifically, TLR9 activation can induce proinflammatory signaling in transformed epithelial cells, but does not elicit oncogene expression or cancer cell proliferation. Conversely, TLR9 ligation induces pancreatic stellate cells (PSCs) to become fibrogenic and secrete chemokines that promote epithelial cell proliferation. TLR9-activated PSCs mediate their protumorigenic effects on the epithelial compartment via CCL11. Additionally, TLR9 has immune-suppressive effects in the tumor microenvironment (TME) via induction of regulatory T cell recruitment and myeloid-derived suppressor cell proliferation. Collectively, our work shows that TLR9 has protumorigenic effects in pancreatic carcinoma which are distinct from its influence in extrapancreatic malignancies and from the mechanistic effects of other TLRs on pancreatic oncogenesis. © 2015 Zambirinis et al.

Lipotoxicity: Effects of Dietary Saturated and Transfatty Acids

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Débora Estadella

2013-01-01

Full Text Available The ingestion of excessive amounts of saturated fatty acids (SFAs and transfatty acids (TFAs is considered to be a risk factor for cardiovascular diseases, insulin resistance, dyslipidemia, and obesity. The focus of this paper was to elucidate the influence of dietary SFA and TFA intake on the promotion of lipotoxicity to the liver and cardiovascular, endothelial, and gut microbiota systems, as well as on insulin resistance and endoplasmic reticulum stress. The saturated and transfatty acids favor a proinflammatory state leading to insulin resistance. These fatty acids can be involved in several inflammatory pathways, contributing to disease progression in chronic inflammation, autoimmunity, allergy, cancer, atherosclerosis, hypertension, and heart hypertrophy as well as other metabolic and degenerative diseases. As a consequence, lipotoxicity may occur in several target organs by direct effects, represented by inflammation pathways, and through indirect effects, including an important alteration in the gut microbiota associated with endotoxemia. Interactions between these pathways may perpetuate a feedback process that exacerbates an inflammatory state. The importance of lifestyle modification, including an improved diet, is recommended as a strategy for treatment of these diseases.

**-Postprandial pancreatic ["1"1C]methionine uptake after pancreaticoduodenectomy mirrors basal beta cell function and insulin release

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Steiner, Emanuel; Kazianka, Lukas; Breuer, Robert; Miholic, Johannes; Hacker, Marcus; Wadsak, Wolfgang; Mitterhauser, Markus; Stimpfl, Thomas; Reiter, Birgit; Karanikas, Georgios

2017-01-01

[S-methyl-"1"1C]-L-methionine (["1"1C]MET) uptake in the pancreas might be a central indicator of beta cell function. Since gastric emptying was recently shown to influence glycemic control in subjects after pancreaticoduodenectomy (PD, the surgical treatment of neoplasms of the pancreas head), we looked for imaginable relationships between gastric emptying, pre- and postprandial insulin concentrations, and ["1"1C]MET uptake. Nineteen tumor-free survivors after PD (age mean ± SD: 61 ± 8.7 yrs.; 10 male, 9 female) and 10 healthy controls (age: 27 ± 8.7 yrs.; 7 male, 3 female) were given a mixed test meal. One gram of paracetamol was ingested with the meal to evaluate the speed of gastric emptying. Insulin, glucose, and paracetamol plasma concentrations were measured before and over 180 minutes after ingestion. Beta cell function was calculated from fasting glucose and insulin plasma concentrations. Simultaneously, 800 MBq of ["1"1C]MET were administered and the activity (maximum tissue standardized uptake values [SUVmax]) over the pancreas was measured at 15, 30, and 60 minutes after injection. Total integrated SUVmax (area under the curve [AUC]) and incremental SUVmax were calculated. The uptake of ["1"1C]MET in the pancreas was significantly higher (p < 0.0001) in controls compared to the PD group. Gastric emptying was significantly slower in controls compared to pancreatectomy subjects (p < 0.0001). Paracetamol AUC_3_0 correlated with the SUVmax increment between 15 and 30 minutes (R"2 = 0.27, p = 0.0263), suggesting a relationship between gastric emptying and the uptake of ["1"1C]MET. Total integrated SUVmax correlated with insulin AUC_6_0 (R"2 = 0.66,p < 0.0001) in patients after PD. Multivariate regression analysis revealed insulin AUC_6_0 and beta cell function, calculated from the fasting insulin to glucose ratio, as independent predictors of "1"1C-methionine uptake, i.e. total integrated SUVmax, in patients after PD (R"2 = 0.78, p < 0.0001). Postprandial

**-Postprandial pancreatic [{sup 11}C]methionine uptake after pancreaticoduodenectomy mirrors basal beta cell function and insulin release

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Steiner, Emanuel; Kazianka, Lukas; Breuer, Robert; Miholic, Johannes [Medical University of Vienna, Department of Surgery, Vienna (Austria); Hacker, Marcus; Wadsak, Wolfgang; Mitterhauser, Markus [Medical University of Vienna, Department of Biomedical Imaging and Image-Guided Therapy, Division of Nuclear Medicine, Vienna (Austria); Stimpfl, Thomas; Reiter, Birgit [Medical University of Vienna, Clinical Institute of Laboratory Medicine, Forensic Toxicology, Vienna (Austria); Karanikas, Georgios [Medical University of Vienna, Department of Biomedical Imaging and Image-Guided Therapy, Division of Nuclear Medicine, Divisional Head PET-PET/CT (Nuclear Medicine), Vienna (Austria)

2017-03-15

[S-methyl-{sup 11}C]-L-methionine ([{sup 11}C]MET) uptake in the pancreas might be a central indicator of beta cell function. Since gastric emptying was recently shown to influence glycemic control in subjects after pancreaticoduodenectomy (PD, the surgical treatment of neoplasms of the pancreas head), we looked for imaginable relationships between gastric emptying, pre- and postprandial insulin concentrations, and [{sup 11}C]MET uptake. Nineteen tumor-free survivors after PD (age mean ± SD: 61 ± 8.7 yrs.; 10 male, 9 female) and 10 healthy controls (age: 27 ± 8.7 yrs.; 7 male, 3 female) were given a mixed test meal. One gram of paracetamol was ingested with the meal to evaluate the speed of gastric emptying. Insulin, glucose, and paracetamol plasma concentrations were measured before and over 180 minutes after ingestion. Beta cell function was calculated from fasting glucose and insulin plasma concentrations. Simultaneously, 800 MBq of [{sup 11}C]MET were administered and the activity (maximum tissue standardized uptake values [SUVmax]) over the pancreas was measured at 15, 30, and 60 minutes after injection. Total integrated SUVmax (area under the curve [AUC]) and incremental SUVmax were calculated. The uptake of [{sup 11}C]MET in the pancreas was significantly higher (p < 0.0001) in controls compared to the PD group. Gastric emptying was significantly slower in controls compared to pancreatectomy subjects (p < 0.0001). Paracetamol AUC{sub 30} correlated with the SUVmax increment between 15 and 30 minutes (R{sup 2} = 0.27, p = 0.0263), suggesting a relationship between gastric emptying and the uptake of [{sup 11}C]MET. Total integrated SUVmax correlated with insulin AUC{sub 60} (R{sup 2} = 0.66,p < 0.0001) in patients after PD. Multivariate regression analysis revealed insulin AUC{sub 60} and beta cell function, calculated from the fasting insulin to glucose ratio, as independent predictors of {sup 11}C-methionine uptake, i.e. total integrated SUVmax, in

Multilayer network representation of membrane potential and cytosolic calcium concentration dynamics in beta cells

International Nuclear Information System (INIS)

Gosak, Marko; Dolenšek, Jurij; Markovič, Rene; Slak Rupnik, Marjan; Marhl, Marko; Stožer, Andraž

2015-01-01

Highlights: • Physiological processes within and among pancreatic beta cells are very complex. • We analyze the simultaneous recordings of membrane potential and calcium dynamics. • We represent the interaction patterns among beta cells as a multilayer network. • The nature of the intracellular dynamics is found to rely on the network structure. - Abstract: Modern theory of networks has been recognized as a very successful methodological concept for the description and analysis of complex systems. However, some complex systems are more complex than others. For instance, several real-life systems are constituted by interdependent subsystems and their elements are subjected to different types of interactions that can also change with time. Recently, the multilayer network formalism has been proposed as a general theoretical framework for the description and analysis of such multi-dimensional complex systems and is acquiring more and more prominence in terms of a new research direction. In the present study, we use this methodology for the description of functional connectivity patterns and signal propagation between pancreatic beta cells in an islet of Langerhans at the levels of membrane potential (MP) and cytosolic calcium concentration ([Ca"2"+]_c) dynamics to study the extent of overlap in the two networks and to clarify whether time lags between the two signals in individual cells are in any way dependent on the role these cells play in the functional networks. The two corresponding network layers are constructed on the basis of signal directions and pairwise correlations, whereas the interlayer connections represent the time lag between both measured signals. Our results confirm our previous finding that both MP and [Ca"2"+]_c change spread across an islet in the form of a depolarization and a [Ca"2"+]_c wave, respectively. Both types of waves follow nearly the same path and the networks in both layers have a similar but not entirely the same structure

[Effects of ezrin silencing on pancreatic cancer cell line Panc-1].

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Meng, Yun-xiao; Yu, Shuang-ni; Lu, Zhao-hui; Chen, Jie

2012-12-01

To explore the effects of ezrin silencing on pancreatic cancer cell line Panc-1. Pancreatic cancer cell line Panc-1 was transfected with ezrin silencing plasmid. The proliferation and the cell cycle status were determined by CCK-8 assay and flow cytometry analysis, respectively. Cellular membrane protrusions/microvilli formation were visualized by scanning election microscopy. Colony formation assay was used to determine the cell anchor-independent growth ability in vitro. Trans-filter migration and invasion assays were performed with 8 µm pore inserts in a 24-well BioCoat chamber with/without Matrigel. Ezrin silencing decreased cellular protrusions/microvilli formation, anchorage-independent growth, cell migration and invasion, but had no effects on cell proliferation in vitro and cell cycle, in pancreatic cancer cell line Panc-1. Ezrin expression affects the cellular protrusions/microvilli formation, anchorage-independent growth, cell migration and invasion in pancreatic cancer cell line Panc-1.

File list: Unc.Pan.50.AllAg.Pancreatic_cancer_cells [Chip-atlas[Archive

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Profile of MMP and TIMP Expression in Human Pancreatic Stellate Cells: Regulation by IL-1α and TGFβ and Implications for Migration of Pancreatic Cancer Cells

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Vegard Tjomsland

2016-07-01

Full Text Available Pancreatic ductal adenocarcinoma is characterized by a prominent fibroinflammatory stroma with both tumor-promoting and tumor-suppressive functions. The pancreatic stellate cell (PSC is the major cellular stromal component and the main producer of extracellular matrix proteins, including collagens, which are degraded by metalloproteinases (MMPs. PSCs interact with cancer cells through various factors, including transforming growth factor (TGFβ and interleukin (IL-1α. The role of TGFβ in the dual nature of tumor stroma, i.e., protumorigenic or tumor suppressive, is not clear. We aimed to investigate the roles of TGFβ and IL-1α in the regulation of MMP profiles in PSCs and the subsequent effects on cancer cell migration. Human PSCs isolated from surgically resected specimens were cultured in the presence of pancreatic cancer cell lines, as well as IL-1α or TGFβ. MMP production and activities in PSCs were quantified by gene array transcripts, mRNA measurements, fluorescence resonance energy transfer–based activity assay, and zymography. PSC-conditioned media and pancreatic cancer cells were included in a collagen matrix cell migration model. We found that production of IL-1α by pancreatic cancer cells induced alterations in MMP and tissue inhibitors of matrix metalloproteinase (TIMP profiles and activities in PSCs, upregulated expression and activation of MMP1 and MMP3, and enhanced migration of pancreatic cancer cells in the collagen matrix model. TGFβ counteracted the effects of IL-1α on PSCs, reestablished PSC MMP and TIMP profiles and activities, and inhibited migration of cancer cells. This suggests that tumor TGFβ has a role as a suppressor of stromal promotion of tumor progression through alterations in PSC MMP profiles with subsequent inhibition of pancreatic cancer cell migration.

Effects of Ghrelin miRNA on Inflammation and Calcium Pathway in Pancreatic Acinar Cells of Acute Pancreatitis.

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Tang, Xiping; Tang, Guodu; Liang, Zhihai; Qin, Mengbin; Fang, Chunyun; Zhang, Luyi

The study investigated the effects of endogenous targeted inhibition of ghrelin gene on inflammation and calcium pathway in an in vitro pancreatic acinar cell model of acute pancreatitis. Lentiviral expression vector against ghrelin gene was constructed and transfected into AR42J cells. The mRNA and protein expression of each gene were detected by reverse transcription polymerase chain reaction, Western blotting, or enzyme-linked immunosorbent assay. The concentration of intracellular calcium ([Ca]i) was determined by calcium fluorescence mark probe combined with laser scanning confocal microscopy. Compared with the control group, cerulein could upregulate mRNA and protein expression of inflammatory factors, calcium pathway, ghrelin, and [Ca]i. mRNA and protein expression of inflammatory factors increased significantly in cells transfected with ghrelin miRNA compared with the other groups. Intracellular calcium and expression of some calcium pathway proteins decreased significantly in cells transfected with ghrelin miRNA compared with the other groups. Targeted inhibition of ghrelin gene in pancreatic acinar cells of acute pancreatitis can upregulate the expression of the intracellular inflammatory factors and alleviate the intracellular calcium overload.

File list: His.Pan.20.AllAg.Pancreatic_cancer_cells [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: His.Pan.10.AllAg.Pancreatic_cancer_cells [Chip-atlas[Archive

Lifescience Database Archive (English)

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